Number Of Cells In G1 Phase Research Articles

Bone marrow mesenchymal stem cells-derived exosomal lncRNA GAS5 mitigates heart failure by inhibiting UL3/Hippo pathway-mediated ferroptosis

BackgroundExosomes (Exos) are involved in the therapeutic effects of bone marrow mesenchymal stem cells (BMSCs) on heart failure (HF). We investigated the molecular mechanisms underlying the involvement of BMSC-Exos in ferroptosis on HF.MethodsA rat model of HF and cellular model of hypoxia were established. BMSC-Exos were injected into model rats or co-cultured with model cells. In model rats, the cardiac function (echocardiography), oxidative stress (commercial kits), pathological damage (HE staining), fibrosis (MASSON staining), iron deposition (Prussian blue staining), and cell apoptosis (TUNEL staining) were examined. Viability (cell counting kit-8; CCK-8), cell cycle (flow cytometry), oxidative stress, and Fe2+ levels were detected in the model cells. GAS5, UL3, YAP, and TAZ expression were detected using qRT-PCR, western blotting, and immunohistochemistry analyses.ResultsBMSC-Exos restored cardiac function and inhibited oxidative stress, apoptosis, pathological damage, fibrosis, and iron deposition in myocardial tissues of HF rats. In hypoxic cells, BMSC-Exos increased cell viability, decreased the number of G1 phase cells, decreased Fe2+ levels, and inhibited oxidative stress. Ferrostatin-1 (a ferroptosis inhibitor) exhibited a synergistic effect with BMSC-Exos. Additionally, GAS5 was upregulated in BMSC-Exos, further upregulating its target UL3 and Hippo pathway effectors (YAP and TAZ). The relieving effects of BMSC-Exos on HF or hypoxia-induced injury were enhanced by GAS5 overexpression, but weakened by UL3 silencing or verteporfin (a YAP inhibitor).ConclusionsGAS5-harbouring BMSC-Exos inhibited ferroptosis by regulating the UL3/Hippo pathway, contributing to HF remission in vivo and in vitro.

Flow cytometry study of DNA transformation dynamics in ВНК-21/SUSP/ARRIAH cell culture during rabies virus reproduction

The study examines the DNA transformation dynamics of ВНК-21/SUSP/ARRIAH subline cells during rabies virus reproduction. Cells infected with the virus and control intact cells were cultivated under similar conditions. The identification of dependence of the virus infectivity on reproduction time revealed that the virus infectivity titre increased from (3.2 ± 0.2) lg CCID50/cm3 at the time of inoculation to (7.63 ± 0.3) lg CCID50/cm3 after 48 hours of reproduction, with the most intensive increase having been observed within the first 24 hours. The cell concentration changed from 0.5 to 1.9 million/cm3, i.e. increased by a factor of 3.8. After 24 hours, the cell growth rate slowed down. Findings from the examination of cell cycle phases during rabies virus reproduction in the host cell allowed for the estimation of duration and predominance of G1, S, G2 + M phases at different stages of cultivation. The dynamics of changes in the apoptotic cell population in the control and test samples was similar within 36 hours of cultivation. After the said period, the proportion of apoptotic infected cells was 28–42% higher than that of apoptotic control cells. After 9 hours, the proportion of cells undergoing G1 phase increased by 11.7% in the test samples, whereas it decreased by 16.6% in the control samples. Subsequently, the number of G1 phase cells in the control and test samples changed in the same way: a 40% decrease was observed after 15–18 hours, it was followed by a 45–46% growth jump, then again a 39–40% decrease and an increase were observed. After 33 hours of reproduction and till the end of cultivation, the proportion of infected cells undergoing G1 phase was significantly higher (by 12–21%) as compared with control cells. The percentage of S phase cells in the test and control samples was the same during the first day of the virus reproduction, with sharp jump-like 3.4- and 2.4-fold increases having been observed after 15 and 24 hours, respectively. After 24 hours, the infected and control cells began to demonstrate differences, which gradually increased from 8 to 137% by the end of reproduction. After 30 hours of reproduction, the proportion of test sample cells undergoing G2 + M phase began to decrease by 17–28% as compared with the control cells. The cell switch-over to the synthesis of complete rabies virus particles occurred after 24 hours of reproduction. This is indicated by changes in the host cell cycle phases, as well as by the slowing down of ВНК-21/SUSP/ARRIAH cell population growth.

Bta-miR-484 Targets SFRP1 and Affects Preadipocytes Proliferation, Differentiation, and Apoptosis.

MicroRNAs (miRNAs) are essential regulators of numerous biological processes in animals, including adipogenesis. Despite the abundance of miRNAs associated with adipogenesis, their exact mechanisms of action remain largely unknown. Our study highlights the role of bta-miR-484 as a major regulator of adipocyte proliferation, apoptosis, and differentiation. Here, we demonstrated that the expression of bta-miR-484 initially increased during adipogenesis before decreasing. Overexpression of bta-miR-484 in adipocytes ultimately inhibited cell proliferation and differentiation, reduced the number of EdU fluorescence-stained cells, increased the number of G1 phase cells, reduced the number of G2 and S phase cells, and downregulated the expression of proliferation markers (CDK2 and PCNA) and differentiation markers (CEBPA, FABP4, and LPL). Additionally, overexpression of bta-miR-484 promoted the expression of apoptosis-related genes (Caspase 3, Caspase 9, and BAX), and increased the number of apoptotic cells observed via flow cytometry. In contrast, bta-miR-484 inhibition in adipocytes yielded opposite effects to those observed during bta-miR-484 overexpression. Moreover, luciferase reporter assays confirmed SFRP1 as a target gene of bta-miR-484, and revealed that bta-miR-484 downregulates SFRP1 mRNA expression. These findings offer compelling evidence that bta-miR-484 targets SFRP1, inhibits proliferation and differentiation, and promotes apoptosis. Therefore, these results offer novel insights into the bta-miR-484 regulation of adipocyte growth and development.

Abstract 4604: Effect of CDK 4/6 inhibition on anti-angiogenesis effect in gastric cancer

Abstract Angiogenesis, a hallmark of cancer, is a phenomenon in which new blood vessels are created from existing blood vessels. As the tumor grows, they secrete ligands such as vascular endothelial growth factor (VEGF). Therefore, anti-tumor effect could be achieved by preventing tumor-associated angiogenesis. In previous reports, CDK4/6 has been acknowledged to regulate VEGF transcription in addition to cell cycle regulation. There are few studies explaining the angiogenesis-related effects of CDK4/6 inhibitors in gastric cancer (GC). In this study, we aim to verify the anti-angiogenesis effect through CDK4/6 inhibition. First, we confirmed proliferation and angiogenesis in endothelial cells. Next, the change in VEGF expression induced by CDK4/6 inhibitor in GC cell lines was investigated. In-vitro angiogenesis research was conducted using human umbilical vein endothelial cells (HUVEC). Abemaciclib was used as a CDK4/6 inhibitor. To determine the efficacy of abemaciclib, HUVEC was treated with abemaciclib for 3 days and cell viability was assessed by CCK-8 assay. IC50 value was determined by the CalcuSyn software. Furthermore, tube formation assay, wound healing assay, cell cycle analysis and SA-β-gal assay were performed to explore effect of abemaciclib on HUVEC. VEGF-A expression was evaluated by ELISA. First, we identified direct anti-angiogenesis effect of abemaciclib on HUVEC. The IC50 value was 1.28 μM. At clinically achievable concentrations of abemaciclib (Cmax, 0.5 μM), HUVEC only showed a 20% growth inhibition. Abemaciclib did not suppress tube formation and migratory potential of HUVECs. However, abemaciclib induced a G1 arrest in HUVEC after 0.5 μM abemaciclib treatment for 24hr. To ensure whether the increase in the number of G1 phase cells is due to senescence, SA-β-gal assay was performed. The levels of SA-β-gal-positive cell staining in abemaciclib-treated group were higher than control, suggesting that abemaciclib induced cell senescence on HUVEC. While our results suggested that CDK4/6 inhibitor did not show direct anti-angiogenic effect on endothelial cells, we studied indirect anti-angiogenesis effect of abemaciclib in GC cell lines through regulation of VEGF transcription. Conditioned media of 49 GC cell lines had a wide range of VEGF secretion ranged from 0.146 to 22.676 ng/mL. (ave. 4.07 ng/mL). We have set the cut off of secreted VEGF as 10 ng/mL, at which angiogenesis can be induced. There were four cell lines (4/49, 8.2%) whose VEGF concentrations were more than the angiogenesis-inducing VEGF concentration (10 ng/mL). Interestingly, 3 of these 4 GC cell lines demonstrated a decrease in VEGF secretion, following 0.5 μM abemaciclib treatment. In conclusion, we did not observe inhibition of angiogenesis in HUVEC. However, CDK4/6 inhibition can cause senescence in HUVEC, inhibit tumor growth, and indirectly induce anti-angiogenic effects in gastric cancer. Citation Format: Sang Woo Cho, Woo Sun Kwon, Tae Soo Kim, Juin Park, Eun Seo Kim, Hyun Cheol Chung, Sun Young Rha. Effect of CDK 4/6 inhibition on anti-angiogenesis effect in gastric cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4604.

Regulatory effect and mechanism of APN gene on porcine epidemic diarrhea virus resistance.

The expression level of aminopeptidase N (APN) is evidently correlated with porcine epidemic diarrhea virus (PEDV) infectivity. This study aims to examine the mechanisms regulating APN expression level in response to PEDV infection. Quantitative real time PCR was performed herein to detect gene expression dynamics at various timepoints after PEDV infection. Subsequently, CRISPR/Cas9 gene editing technology was used to generate a APN-knockout IPEC-J2 cell line, exploring the effects of APN on cell proliferation by propidium iodide staining and anti-PEDV activity by indirect immunofluorescence assay. Ultimately, the effects of single nucleotide polymorphisms (SNPs) and methylation in the APN promoter region on gene expression were analyzed by using bisulfite sequencing PCR and dual luciferase reporter gene assay. APN expression was significantly upregulated within 4-24h post-infection. The cytoactivity of the APN-knockout IPEC-J2 cell line was markedly suppressed at different timepoints. Further, cell cycle analyses indicated an increase in the number of G1-phase cells and a significant decrease in that of S-phase cells. Moreover, key cyclical factors regulating the G1 phase were highly expressed in APN-knockout cells. The RNA copies of viral particles and mRNA levels of antiviral genes and inflammatory cytokines in APN-knockout cells were markedly decreased within 24h of PEDV infection. Similarly, indirect immunofluorescence assay confirmed that the number of PEDV particles was significantly decreased. Sequence analysis revealed two CpG islands in the APN promoter region. However, there was no evident correlation between the methylation status of APN promoter and mRNA levels. Dual luciferase reporter gene assay showed that the SNP rs326030589 (G/A) significantly increased the promoter activity of APN. These results suggested that APN knockout enhanced the resistance of IPEC-J2 cells to PEDV. Moreover, rs326030589 in the APN promoter region participated in gene transcription regulation. Our results provide a reference for studying the mechanisms regulating APN and may contribute to the application of APN gene in resistance breeding of swine epidemic diarrhea.

Vobtusine from Voacanga foetida (Blume) Rolfe Induces Apoptosis via Activation of Caspase Pathway in Human HL-60 Leukemia Cell Line

Vobtusine is an aspidosperma-aspidosperma alkaloid isolated from alkaloid DCM base fraction of the bark of Voacanga foetida (Blume) Rolfe (Apocynaceae). In this study, the effect of vobtusine on the cell cycle, apoptosis induction, and Bcl-2 family protein expression were investigated by flow cytometr, DNA fragmentation analysis, and western blotting, respectively. The results of cell cycle analysis indicated the ratio of the number of cells in each phase did not have significant differences depend on vobtusine concentration although cell number in G1 phase had tendency to decrease according to the increasing of vobtusine concentration. Besides, the sub-G1 phase population of HL-60 cells treated with vobtusine was increased compared with that of cells without treatments (5.9%-23.8%). DNA fragmentation was observed from 20 µM, and the degree of fragmentation was dependent on vobtusine concentration. Caspase-3 activity increased 4.6 times compared to control. After being treated with caspase-9 inhibitor, vobtusine-induced elevation of caspase-3 activity decreased. This shows that caspase-3 activity depends on caspase-9. Vobtusine was also induced apoptotic cell death involved a mitochondrial by Bid activation and Bcl-xL downregulation. Therefore, vobtsusine-induced apoptosis process was initiated by caspase-9 via change of Bcl-2 family protein expression and executed by caspase-3, followed by cell death due to their proteolytic activity. These results indicated the mechanism action of vobtusine as anti-cancer compound via intrinsic pathway.

MicroRNA-125a-3p overexpression promotes liver regeneration through targeting proline-rich acidic protein 1

Introduction and objectivesLiver regeneration plays a valuable significance for hepatectomies, and is mainly attributed to hepatocyte proliferation. MicroRNA-125a-3p was reported to be highly associated with liver regeneration process. We studied the underlying mechanism of the functional role of miR-125a-3p in liver regeneration. Materials and methodsThe miR-125a-3p mimics and inhibitor vector were constructed and transfected into primary human liver HL-7702 cells, the transfected cell viability was detected using cell counting kit-8 (CCK-8). Cell cycle distribution was analyzed by flow cytometry. With Targetscan and OUGene prediction, the potential targets of miR-125 were verified by real-time quantitative PCR (qPCR) and luciferase reporter assays in turn. The overexpression vector of proline-rich acidic protein 1 (PRAP1) was constructed and co-transfected with miR-125a-3p mimics into HL-7702 cells, detecting the changes of proliferative capacity and cell cycle distribution. Western blot and qPCR performed to analyze gene expressions. ResultsOverexpressed miR-125a-3p notably increased the hepatocyte viability at 48h, and decreased the number of G1 phase cells (p<0.05). However, miR-125a-3p inhibition suppressed the development of hepatocytes. PRAP1 was the target of miR-125a-3p. After co-transfection with PRAP1 vector, hepatocyte viability was decrease and the G1 phase cell number was increased (p<0.05). More importantly, overexpressed PRAP1 notably decreased the mRNA and protein levels of cyclin D1, cyclin-dependent kinase 2 (CDK2) and cell division cycle 25A (CDC25A). ConclusionThe elevated miR-125a-3p positively correlated with hepatocyte viability and cell cycle progression due to the modulation of PRAP1, and miR-125a-3p may contribute to improving liver regeneration.

Retracted: Effects of microRNA-494 on proliferation, migration, invasion, and apoptosis of medulloblastoma cells by mediating c-myc through the p38 MAPK signaling pathway.

Medulloblastoma (MB) is the most prevalent brain tumor that occurs during childhood and originates from cerebellar granule cell precursors. Based on recent studies, the differential expression of several microRNAs is involved in MB, while the role of microRNA-494 (miR-494) in MB remains unclear. Therefore, we conducted this study to investigate the regulative role of miR-494 in MB cells via the p38 mitogen-activated protein kinase (MAPK) signaling pathway by mediating c-myc. In the current study, MB cells were collected and transfected with miR-494 mimic, miR-494 inhibitor, siRNA- c-myc, and miR-494 inhibitor + siRNA-c-myc. The expressions of miR-494, c-myc, p38 MAPK, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), interleukin-6 (IL-6), metadherin (MTDH), phosphatase and tensin homolog (PTEN) and survivin were determined. Cell proliferation, cell-cycle distribution, apoptosis, migration, and invasion were evaluated. The results revealed that there was a poor expression of miR-494 and high expression of c-myc in MB tissues. C-myc was determined as the target gene of miR-494. In response to miR-494 mimic, MB cells were found to have increased Bax and PTEN expressions, as well as cell number in G1 phase and cell apoptosis and decreased c-myc, p38 MAPK, Bcl-2, MTDH, IL-6, and survivin expression and cell number count in the S phase, cell proliferation, migration, and invasion. In conclusion, the results demonstrated that the upregulation of miR-494 results in the suppression of cell proliferation, migration, and invasion, while it promotes apoptosis of MB cells through the negative mediation of c-myc, which in turn inactivates the p38 MAPK pathway.

Recurrent heat shock impairs the proliferation and differentiation of C2C12 myoblasts.

Heat-related illness and injury are becoming a growing safety concern for the farmers, construction workers, miners, firefighters, manufacturing workers, and other outdoor workforces who are exposed to heat stress in their routine lives. A primary response by a cell to an acute heat shock (HS) exposure is the induction of heat-shock proteins (HSPs), which chaperone and facilitate cellular protein folding and remodeling processes. While acute HS is well studied, the effect of repeated bouts of hyperthermia and the sustained production of HSPs in the myoblast-myotube model system of C2C12 cells are poorly characterized. In C2C12 myoblasts, we found that robust HS (43°C, dose/time) significantly decreased the proliferation by 50% as early as on day 1 and maintained at the same level on days 2 and 3 of HS. This was accompanied by an accumulation of cells at G2 phase with reduced cell number in G1 phase indicating cell cycle arrest. FACS analysis indicates that there was no apparent change in apoptosis (markers) and cell death upon repeated HS. Immunoblot analysis and qPCR demonstrated a significant increase in the baseline expression of HSP25, 70, and 90 (among others) in cells after a single HS (43°C) for 60min as a typical HS response. Importantly, the repeated HS for 60min each on days 2 and 3 maintained the elevated levels of HSPs compared to the control cells. Further, the continuous HS exposure resulted in significant inhibition of the differentiation of C2C12 myocytes to myotubes and only 1/10th of the cells underwent differentiation in HS relative to control. This was associated with significantly higher levels of HSPs and reduced expression of myogenin and Myh2 (P<0.05), the genes involved in the differentiation process. Finally, the cell migration (scratch) assay indicated that the wound closure was significantly delayed in HS cells relative to the control cells. Overall, these results suggest that a repeated HS may perturb the active process of proliferation, motility, and differentiation processes in an in vitro murine myoblast-myotube model.

Serine peptidase inhibitor Kazal type I (SPINK1) promotes BRL-3A cell proliferation via p38, ERK, and JNK pathways.

Serine peptidase inhibitor Kazal type I (SPINK1) has the similar spatial structure as epidermal growth factor (EGF); EGF can interact with epidermal growth factor receptor (EGFR) to promote proliferation in different cell types. However, whether SPINK1 can interact with EGFR and further regulate the proliferation of hepatocytes in liver regeneration remains largely unknown. In this study, we investigated the role of SPINK1 in a rat liver hepatocyte line of BRL-3A in vitro. The results showed the upregulation of endogenous Spink1 (gene addition) significantly increased not only the cell viability, cell numbers in S and G2 /M phase, but also upregulated the genes/proteins expression related to cell proliferation and anti-apoptosis in BRL-3A. In contrast, the cell number in G1 phase and the expression of pro-apoptosis-related genes/proteins were significantly decreased. The similar results were observed when the cells were treated with exogenous rat recombinant SPINK1. Immunoblotting suggested SPINK1 can interact with EGFR. By Ingenuity Pathway Analysis software, the SPINK1 signalling pathway was built; the predicted read outs were validated by qRT-PCR and western blot; and the results showed that p38, ERK, and JNK pathways-related genes/proteins were involved in the cell proliferation upon the treatment of endogenous Spink1 and exogenous SPINK1. Collectively, SPINK1 can associate with EGFR to promote the expression of cell proliferation-related and anti-apoptosis-related genes/proteins; inhibit the expression of pro-apoptosis-related genes/proteins via p38, ERK, and JNK pathways; and consequently promote the proliferation of BRL-3A cells. For the first time, we demonstrated that SPINK1 can associate with EGFR to promote the proliferation of BRL-3A cells via p38, ERK, and JNK pathways. This work has direct implications on the underlying mechanism of SPINK1 in regulating hepatocytes proliferation in vivo and liver regeneration after partial hepatectomy.

Induction of endothelial cell proliferation and von Willebrand factor expression and secretion by leukemic plasma of patients with chronic lymphocytic leukemia before and after inhibition of NF-κB.

Although certain evidence has indicated a role for angiogenesis in the pathophysiology of hematopoietic malignancies, its role in chronic lymphocytic leukemia (CLL) prognosis is yet to be defined. To our knowledge, the effects of CLL plasma on cell culture have not been addressed. Therefore, we investigated the effects of CLL plasma on cell cycle regulation and von Willebrand factor (vWF) secretion, and expression in human umbilical vein endothelial cell cultures (HUVECs). Since nuclear factor-kappa B (NF-κB) transcription factor has been a therapeutic target for treatment of cancer, we inhibited NF-κB using small interfering RNA to clarify if there is a role for this factor in probable effects. The cells were treated with the plasma of patients with CLL. Subsequently, cell cycle phase distribution, vWF secretion, expression, and storage were detected using ELISA, flow cytometry, and immunohistochemical staining. In addition, NF-κB was inhibited using small interfering RNA. Plasma treatment promoted cell cycle progression by decreasing the cell number in G1 phase, while increasing the cell number in S phase and G2M phase. A significant increase of vWF expression, secretion, and storage was found, associated with the vWF levels of patients' plasma. We found that induction of cell cycle promotion, but not vWF expression and secretion, was partially suppressed by this inhibition. We found that endothelial cell cycle and vWF expression and secretion affected by CLL plasma and NF-κB play a role in the former. These findings would be useful for understanding the prognostic importance of plasma angiogenic factor levels in CLL.

Effects of soluble programmed death ligand 1 on regulating the proliferation of T lymphocytes and its mechanism

To explore the effects of soluble programmed death ligand 1 (sPD-L1) on the proliferation of T lymphocytes and its mechanism. T lymphocytes were isolated from healthy human peripheral blood and activated by phytohemagglutinin (PHA). The experiment had group A: resting T lymphocytes, group B: activated T lymphocytes, group C: activated T lymphocytes+sPD-L1Ig, group D: activated T lymphocytes+sPD-L1Ig+membrance-bound immunoglobulin (mIgG) and group E: activated T lymphocytes+sPD-L1Ig+anti-PD-L1 antibody (2H11). The absorbance value (A) of T lymphocytes in each group was measured by cell counting kit (CCK-8). The cell cycle and apoptosis of T lymphocytes induced by sPD-L1 were measured by flow cytometry. And the phosphorylation level of programmed death 1 (PD-1) signaling motif tyrosine was measured by Western blot. Furthermore, the amounts of signal adaptor molecule Src homology 2 domain-containing tyrosine phosphatase (SHP)-1 and SHP-2 were quantified by immunoprecipitation. And the exciting mechanism of sPD-L1 was explored for PD-1 inhibitory signals. CCK-8 study showed that A values in each group were 0.42 ± 0.03, 1.20 ± 0.06, 0.87 ± 0.05, 0.78 ± 0.05 and 1.11 ± 0.09 respectively when the concentration of sPD-L1Ig was 250 ng/ml. The proliferation of T lymphocytes in group C significantly decreased compared with group B (t = 3.946, P = 0.017) while group E significantly increased compared with group D (t = 3.139, P = 0.035). The percentage of cell number in G1 phase of the above-mentioned 5 groups were (94.49 ± 0.50)%, (79.22 ± 0.50)%, (89.62 ± 0.33)%, (92.89 ± 0.80)% and (87.94 ± 0.87)% respectively and group C significantly increased compared with group B (t = 17.310, P < 0.001). The apoptotic rate of the above-mentioned five groups were (35.77 ± 1.82)%, (35.20 ± 2.70)%, (62.77 ± 0.24)%, (64.47 ± 0.44)% and (36.80 ± 3.53)% respectively. And apoptotic rate in group C significantly increased compared with group B (t = 10.160, P = 0.001) while group E significantly decreased compared with group D (t = 7.790, P = 0.002). The expressions of SHP-1 and SHP-2 showed no inter-group difference (all P > 0.05). However, the expressions of p-SHP-1 and p-SHP-2 in group C was higher than those in group B (t = 10.790, P < 0.001; t = 13.051, P < 0.001) while the expression of p-SHP-1 decreased in group E compared with group D (t = 3.361, P = 0.028). Soluble PD-L1 can effectively inhibit the proliferation of T lymphocytes. The phosphorylation of SHP-1 and SHP-2 contributes to the inhibitory signaling of PD-1/sPD-L1 pathway. And anti-PD-L1 blocking antibody may partially restore the proliferation of T lymphocytes through a down-regulated expression of p-SHP-1..

Novel taspine derivative 12k inhibits cell growth and induces apoptosis in lung cell carcinoma

Taspine is an active compound in anticancer agent development. 12k was synthesized with taspine as lead compound bearing biphenyl scaffold and showed potent anticancer activity. Here, we investigated the effect of taspine derivative 12k on A549 lung cells. We showed that 12k not only decreased significantly A549 cell viability, A549 cell colony formation but also impaired A549 cell migration. Moreover, 12k treatment blocked cell cycle progression by increasing cell number in S phase to 42.80% for 6μmol/L vs. 28.86% for control while decreasing cell number in G1 phase. Accordingly, this was associated with an increase protein expression of cyclin E and a decrease protein expression of cyclin D1, cyclin B1 and its associated CDK1 (cdc2). Meanwhile, we found that 12k induced A549 cell apoptosis, which was closely associated with the effect of the Bcl-2 family. Increase of Bad, Bak and Bax expression levels, decrease of Bcl-2 and Mcl-1 expression levels were observed. SiRNA knockdown of c-myc in A549 cells significantly attenuated tumor inhibition effects of 12k. In conclusion, our results demonstrate that 12k has an inhibitory effect on growth of A549 cell by inducing cell cycle arrest and apoptosis.

Zac1 Regulates Cell Cycle Arrest in Neuronal Progenitors via Tcf4

Imprinted genes play a critical role in brain development and mental health, although the underlying molecular and cellular mechanisms remain incompletely understood. The family of basic helix-loop-helix (bHLH) proteins directs the proliferation, differentiation, and specification of distinct neuronal progenitor populations. Here, we identified the bHLH factor gene Tcf4 as a direct target gene of Zac1/Plagl1, a maternally imprinted transcriptional regulator, during early neurogenesis. Zac1 and Tcf4 expression levels concomitantly increased during neuronal progenitor differentiation; moreover, Zac1 interacts with two cis-regulatory elements in the Tcf4 gene locus, and these elements together confer synergistic activation of the Tcf4 gene. Tcf4 upregulation enhances the expression of the cyclin-dependent kinase inhibitor gene p57(Kip2), a paternally imprinted Tcf4 target gene, and increases the number of cells in G1 phase. Overall, we show that Zac1 controls cell cycle arrest function in neuronal progenitors through induction of p57(Kip2) via Tcf4 and provide evidence for cooperation between imprinted genes and a bHLH factor in early neurodevelopment.

Reg4‐induced mitogenesis involves Akt‐GSK3β‐β‐Catenin‐TCF‐4 signaling in human colorectal cancer

Upregulation of regenerating gene 4 (Reg4) is observed in many human gastrointestinal malignancies including colorectal cancer (CRC). We previously reported a Reg4-mediated induction of epidermal growth factor receptor-Akt-AP1 signaling regulating CRC cell apoptosis. However, the role of Reg4 in the regulation of CRC cell division is poorly understood. This study tests the hypothesis that Reg4 induces Akt-GSK3β-β-Catenin-TCF-4 signaling to regulate CRC cell division. In vitro models of human CRC were used to determine the role of Reg4 in regulation of CRC cell division. Cell cycle studies demonstrated that Reg4 treatment significantly decreased CRC cell number in G1 phase and increased in G2 phase. Subsequently Reg4 significantly increased the mitotic index of CRC cells. As assessed by real-time RT-PCR and Western blot analyses, Reg4 significantly increased the expression of cell cycle regulatory genes Cyclin D1 and D3, and associated Cyclin-dependent kinases (CDK4 and CDK6). Reg4-mediated increase in these genes involved a pathway that included an induced Akt activity by increasing phosphorylation of Thr308 and Ser473, a reduced glycogen synthase kinase 3β (GSK-3β) activity by increasing phosphorylation of Ser9, an induced nuclear translocation of β-Catenin by decreasing phosphorylation of Ser33/37/Thr41, and an increased TCF-4 transcriptional activity. Furthermore, antagonism of Reg4-signaling using Reg4-specific mAbs (2H6 and 3E5) and Akt inhibitor significantly decreased, whereas agonism using GSK-3β antagonist (SB216763) significantly increased mitotic index and proliferation of CRC cells. These results identify Reg4 as a key regulator of the CRC cell division and proliferation, hence a potential target of human CRC treatment.

Alpha-ketoglutarate (AKG) inhibits proliferation of colon adenocarcinoma cells in normoxic conditions

Background and objective. Alpha-ketoglutarate (AKG), a key intermediate in Krebs cycle, is an important biological compound involved in the formation of amino acids, nitrogen transport, and oxidation reactions. AKG is already commercially available as a dietary supplement and its supplementation with glutamine, arginine, or ornithine alpha-ketoglutarate has been recently considered to improve anticancer immune functions. It is well documented that AKG treatment of Hep3B hepatoma cells in hypoxia induced HIF-alpha (hypoxia-inducible factor) degradation and reduced vascular endothelial growth factor (VEGF) synthesis. Moreover, AKG showed potent antitumor effects in murine tumor xenograft model, inhibiting tumor growth, angiogenesis, and VEGF gene expression. However, the mechanisms of its anticancer activity in normoxia have not been examined so far. Results. Here, we report that in normoxia, AKG inhibited proliferation of colon adenocarcinoma cell lines: Caco-2, HT-29, and LS-180, representing different stages of colon carcinogenesis. Furthermore, AKG influenced the cell cycle, enhancing the expression of the inhibitors of cyclin-dependent kinases p21 Waf1/Cip1 and p27 Kip1. Moreover, expression of cyclin D1, required in G1/S transmission, was decreased, which accompanied with the significant increase in cell number in G1 phase. AKG affected also one the key cell cycle regulator, Rb, and reduced its activation status. Conclusion. In this study for the first time, the antiproliferative activity of AKG on colon adenocarcinoma Caco-2, HT-29, and LS-180 cells in normoxic conditions was revealed. Taking into consideration an anticancer activity both in hypoxic and normoxic conditions, AKG may be considered as a new potent chemopreventive agent.

Drosha regulates hMSCs cell cycle progression through a miRNA independent mechanism

Recently we demonstrated that the miRNA regulate human mesenchymal stem cells (hMSCs) differentiation. To determine the role of the miRNA pathway in hMSCs proliferation, Drosha and Dicer knockdown hMSCs were generated using a lentiviral based tetracycline inducible shRNA. hMSCs with reduced Drosha expression had a significantly reduced proliferation rate, while hMSCs with reduced Dicer expression displayed a proliferation rate similar to untransduced cells. Cell cycle analysis identified that unlike Dicer knockdown, Drosha knockdown hMSCs contained an increased number of G1 phase cells, with a reduced level of cells in S phase, compared to controls. ELISAs of hMSCs revealed decreased levels of pRB and stable levels of total RB with Drosha knockdown. Two key regulators of the G1/S phase transition, cyclin dependent kinase inhibitor 2A (p16) and cyclin dependent kinase inhibitor 2B (p15), were increased in Drosha knockdown cells but not in Dicer knockdown. Transcripts of 28S and 18S rRNA were significantly reduced in Drosha knockdown hMSCs, with no change in rRNA levels in Dicer knockdown hMSCs. 45S pre-rRNA transcripts were not significantly different in either knockdown model. The above results indicate that Drosha modifies hMSCs proliferation through a miRNA independent mechanism, potentially by regulating rRNA processing.

Effects of receptor activator of NF-κB ligand gene silencing on the human osteoblast-like MG63 cells

The osteoprotegerin (OPG)/receptor activator of nuclear factor-κB ligand (RANKL)/receptor activator of NF-κB (RANK) system plays an important role in the pathogenesis of metabolic bone diseases. This study is aimed to investigate effects and mechanisms of RANKL gene silencing on the function of human osteoblast-like MG63 cells by RNA interference using a lentivirus-based small hairpin RNA (vshRNA) delivery system. After RANKL-specific vshRNAs were designed, constructed and transfected into MG63 cells, changes in the expression levels of RANKL mRNA and protein were determined by Western blot and RT-PCR, respectively; changes in cell activity and cell cycle distribution were examined by thiazolyl blue tetrazolium bromide assay and flow cytometry. The expression levels of RANKL mRNA and protein in MG63 cells were reduced by transferring RANKL-specific vshRNAs. Compared to cells infected with negative control virus, the proliferation of cells infected with the recombinant virus was more likely to be inhibited. Furthermore, the cell cycle of MG63 was altered, with the number of G1 phase cells decreasing significantly (P < 0.05). RANKL-specific vshRNAs can significantly inhibit the expression of the target gene in MG63 cells. RANKL gene silencing can inhibit the proliferation and alter the cell cycle of MG63 cells. Our findings suggest that RANKL might play an important role in the regulation of growth and cell cycle of MG63 cells.

Human respiratory syncytial virus reduces the number of cells in S-phase and increases GADD153 expression in HEp-2 cells

Human respiratory syncytial virus (HRSV) associated with bronchiolitis and asthma is known to replicate actively in ciliated epithelial cells. However, little is known about the influence of HRSV replication on the cell cycle. We found that HRSV infection of HEp-2 cells led to a reduction of the number of cells in S-phase, to an increase in the number of cells in G1-phase, together with an increase of GADD153 mRNA levels and GADD153 protein expression. These results implied that a shorter S-phase supported HRSV replication suggesting possible strategies for interfering with productive HRSV infection.

Hyperoxia inhibits several critical aspects of vascular development

Normal human retinal vascular development uses angiogenesis and vasculogenesis, both of which are interrupted in the vaso-obliteration phase of retinopathy of prematurity (ROP). Canine oxygen-induced retinopathy (OIR) closely resembles human ROP. Canine retinal endothelial cells (ECs) and angioblasts were used to model OIR and characterize the effects of hyperoxia on angiogenesis and vasculogenesis. Cell cycle analysis showed that hyperoxia reduced the number of G1 phase cells and showed increased arrest in S phase for both cell types. Migration of ECs was significantly inhibited in hyperoxia (P < 0.01). Hyperoxia disrupted the cytoskeleton of angioblasts but not ECs after 2 days. Differentiation of angioblasts into ECs (determined by acetylated low-density lipoprotein uptake) was evaluated after basic fibroblast growth factor treatment. Differentiation of angioblasts into pericytes was determined by smooth muscle actin expression after treatment with platelet-derived growth factor. Differentiation into ECs was significantly inhibited by hyperoxia (P < 0.0001). The percentage of CXCR4(+) cells (a marker for retinal vascular precursors) increased in both treatment groups after hyperoxia. These data show novel mechanisms of hyperoxia-induced disruption of vascular development.