Helicobacter pylori inhibits gastric cell cycle progression

Helicobacter pylori infection of the gastric mucosa is associated with changes in gastric epithelial cell proliferation. In vitro studies have shown that exposure to H. pylori inhibits proliferation of gastric cells. This study sought to investigate the cell cycle progression of gastric epithelial cell lines in the presence and absence of H. pylori. Unsynchronized and synchronized gastric epithelial cell lines AGS and KatoIII were exposed to H. pylori over a 24-h period. Cell cycle progression was determined by flow cytometry using propidium iodide (PI), and by analysis of cyclin E, p21, and p53 protein expression using Western blots. In the absence of H. pylori 40, 45, and 15% of unsynchronized AGS cells were in G(0)-G(1), S, and G(2)-M phases, respectively, by flow cytometry analysis. When AGS cells were cultured in the presence of H. pylori, the S phase decreased 10% and the G(0)-G(1) phase increased 17% after 24 h compared with the controls. KatoIII cells, which have a deleted p53 gene, showed little or no response to H. pylori. When G1/S synchronized AGS cells were incubated with media containing H. pylori, the G(1) phase increased significantly (25%, P < 0.05) compared with controls after 24 h. In contrast, the control cells were able to pass through S phase. The inhibitory effects of H. pylori on the cell cycle of AGS cells were associated with a significant increase in p53 and p21 expression after 24 h. The expression of cyclin E was downregulated in AGS cells following exposure of AGS cells to H. pylori for 24 h. This study shows that H. pylori-induced growth inhibition in vitro is predominantly at the G(0)-G(1) checkpoint. Our results suggest that p53 may be important in H. pylori-induced cell cycle arrest. These results support a role for cyclin-dependent kinase inhibitors in the G(1) cell cycle arrest exerted by H. pylori and its involvement in changing the regulatory proteins, p53, p21, and cyclin E in the cell cycle.     

Activation of muscarinic acetylcholine receptors inhibits cell cycle progression of small cell lung carcinoma.

We previously reported that activation of muscarinic acetylcholine receptors (mAChR) of M3 subtype causes hydrolysis of phosphoinositides and inhibits voltage-gated Ca2+ channel activity in small cell lung carcinoma (SCLC) cells. We now report that mAChR activation causes exponentially growing SCLC cells to arrest in S and G2/M phases of the cell cycle, concomitant with a decrease in DNA synthesis. Cell cycle progression and DNA synthesis resume when mAChR are down-regulated. In serum-starved SCLC cells, mAChR activation inhibits DNA synthesis induced by serum, bombesin, insulin, or insulin-like growth factor-I. The finding that DNA synthesis is inhibited even when mAChR are activated after exposure of cells to growth factors indicates that decreased signal transduction by growth factor receptors is not the mechanism of mAChR-mediated growth inhibition. Our data suggest that mAChR activation disrupts a common event that is induced by different growth factors and is fundamental for cell cycle progression.     

Indomethacin inhibits cell growth of medullary thyroid carcinoma by reducing cell cycle progression into S phase

Indomethacin, a non-steroidal anti-inflammatory drug (NSAID), has been reported to inhibit the growth of medullary thyroid carcinoma (MTC) cells in vitro. However, the mechanism of inhibition of MTC cell growth by indomethacin and its potency have yet to be revealed. We examined the effect of indomethacin on three different MTC cell lines (TT cells, DRO 81-1 cells and HRO 85-1 cells) and two non-MTC cells. The mechanism of indomethacin action in MTC cells was investigated by analyzing intracellular prostaglandin level, apoptosis, and cell cycle in TT cells. Indomethacin inhibited cell growth of all three MTC cell lines but not normal thyroid cells or anaplastic thyroid carcinoma cells. Indomethacin at 10 microM or greater showed a dose response inhibition of cell growth. Indomethacin at 25 muM, a putative therapeutic serum indomethacin level, showed potency similar to 100 to 200 nM sunitinib, a receptor tyrosine kinase inhibitor. To examine whether prostaglandin depletion might determine the inhibition of MTC cell growth, we created different prostaglandin E2 (PGE2) levels in TT cells using three different NSAIDs. A profound PGE2 depletion by indomethacin-ester, a potent cyclooxygenase (COX) II inhibitor, showed the least inhibition of cell growth. Indomethacin did not increase apoptosis of TT cells. Indomethacin, but not naproxen or indomethacin-ester, reduced cell cycle progression into S phase; this was unrelated to the degree of PGE2 depletion. The expression of phosphorylated retinoblastoma (pRb) protein that shifts cells from G(1) to S phase was reduced after exposure to indomethacin. In conclusion, indomethacin has specific anti-tumor effect on MTC cells, probably by reducing cell cycle progression into S phase rather than by prostaglandin depletion. Since no drug therapy is currently available for MTC, indomethacin may be one of the therapeutic candidates.     

M2 receptor activation inhibits cell cycle progression and survival in human glioblastoma cells

Muscarinic receptors, expressed in several primary and metastatic tumours, appear to be implicated in their growth and propagation. In this work we have demonstrated that M2 muscarinic receptors are expressed in glioblastoma human specimens and in glioblastoma cell lines. Moreover, we have characterized the effects of the M2 agonist arecaidine on cell growth and survival both in two different glioblastoma cell lines (U251MG and U87MG) and in primary cultures obtained from different human biopsies. Cell growth analysis has demonstrated that the M2 agonist arecaidine strongly decreased cell proliferation in both glioma cell lines and primary cultures. This effect was dose and time dependent. FACS analysis has confirmed cell cycle arrest at G1/S and at G2/M phase in U87 cells and U251 respectively. Cell viability analysis has also shown that arecaidine induced severe apoptosis, especially in U251 cells. Chemosensitivity assays have, moreover, shown arecaidine and temozolomide similar effects on glioma cell lines, although IC50 value for arecaidine was significantly lower than temozolomide. In conclusion, we report for the first time that M2 receptor activation has a relevant role in the inhibition of glioma cell growth and survival, suggesting that M2 may be a new interesting therapeutic target to investigate for glioblastoma therapy.     

Wogonin inhibits cell cycle progression by activating the glycogen synthase kinase-3 beta in hepatocellular carcinoma

BackgroundWogonin has been reported to exhibit various biological activities such as anti-inflammation, anti-microbial, and anti-tumor. Previous studies have demonstrated that wogonin could down-regulate Cyclin D1 activity on multiple cancers. However, the related mechanisms have not been fully elucidated so far.PurposeThe aim of the current study was to explore whether wogonin can suppress hepatocellular carcinoma (HCC) progression and the mechanism of wogonin in inhibiting Cyclin D1 expression.MethodsHerein, we assessed the anti-tumor activity of wogonin against hepatocellular carcinoma (HCC) by MTT assay, clonogenic assay, cell cycle analysis and orthotopic xenograft mouse models. Western blot, immunofluoscence assay, co-immunoprecipitation assay, docking program, surface plasmon resonance, site-directed mutagenesis assay and immunohistochemical assay were performed for exploring the underlying mechanisms of wogonin-induced growth inhibition in HCC.ResultsOur results showed that non-toxic dosage of wogonin (10, 20 µM) could inhibit cells proliferation and suppress cells cycle progression in MHCC97L and HepG2 cell. Moreover, the findings from the western blot and immunofluoscence assay confirmed the inhibition action of wogonin (10, 20 µM) on Cyclin D1 expression in MHCC97L cells, and wogonin (10, 20 µM) pre-treatment was capable of promoting Cyclin D1 ubiquitination and degradation in MHCC97L cell. In addition, wogonin promoted phosphorylation of Cyclin D1 on threonine-286 site, the mutation of threonine-286 to alanine-286A blocked Cyclin D1 proteolysis induced by wogonin. Wogonin-promoted Cyclin D1 phosphorylation and subsequent proteolysis may associate with the activation of GSK3beta in cancer cells. The phosphorylated form of GSK3beta (active form) expression was significantly increased after wogonin (20 µM) exposure. Molecular docking study and Biacore SPR analysis of GSK3beta mutant further validated the high-affinity wogonin binding site on GSK3beta. Moreover, in vivo studies further confirmed that phospho-GSK3beta Tyr216 was over-expressed in HCC specimens after wogonin treatment while the amount of Cyclin D1 was significantly decreased.ConclusionIn summary, our data reveal a novel molecular mechanism by which wogonin induces HCC cells cycle arrest and suppresses tumor proliferation.     

LINC00261 elevation inhibits angiogenesis and cell cycle progression of pancreatic cancer cells by upregulating SCP2 via targeting FOXP3

Long non-coding RNAs (lncRNAs) biological functions and molecular mechanisms associated with pancreatic cancer (PC) remain to be poorly elucidated. We aimed to clarify the role of lncRNA LINC00261 (LINC00261) in PC and confirm its regulatory mechanisms. Bioinformatics analysis, RNA pull-down and RIP assays were performed to investigate relationship between LINC00261 and forkhead box P3 (FOXP3). Further, dual-luciferase reporter gene and ChIP assays were employed to confirm the relationship among LINC00261, FOXP3 and sterol carrier protein-2 (SCP2). PC cells were introduced with a series of vectors to verify the effects of LINC00261 and SCP2 on the viability, cell cycle progression, migration and angiogenesis of PC cells. Nude mice with the xenograft tumour were used to evaluate the effects LINC00261 on the tumourigenicity. LINC00261 was lowly expressed in PC tissues and cells. SCP2 was inhibited by LINC00261 through FOXP3. Functionally, upregulated LINC00261 or downregulated SCP2 led to reduced cell viability, migration, angiogenesis and tumourigenicity potentials. This study demonstrated the inhibitory role of LINC00261 in the angiogenesis and cell cycle progression of PC cells. It acts through the negative regulation of SCP2 via targeting FOXP3. Findings in this study highlight a potentially biomarker for PC treatment.     

Endoplasmic reticulum stress inhibits cell cycle progression via induction of p27 in melanoma cells

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), a stress signaling pathway. The UPR coordinates the induction of ER chaperones with decreased protein synthesis and growth arrest in G1 phase of the cell cycle. However, the molecular mechanism underlying UPR-induced G1 cell cycle arrest remains largely unknown. Here we report that activation of the UPR response by tunicamycin (TM), an ER stress inducer, leads to accumulation of p27 and G1 cell cycle arrest in melanoma cells. This accumulation of p27 is due to the inhibition on its polyubiquitination and subsequent degradation upon TM treatment. Correlated with p27 stabilization, the levels of Skp2, an E3 ligase for p27, are decreased in response to TM treatment. More importantly, knockdown of p27 greatly reduces TM-induced G1 cell cycle arrest. Taken together, these data implicate p27 as a critical mediator of ER stress-induced growth arrest.     

Soluble HLA-G inhibits cell cycle progression in human alloreactive T lymphocytes

HLA-G is involved in regulating T cell responses. Various mechanisms have been proposed to explain the inhibition of T cell proliferation. In this context, the possible role of HLA-G in cell cycle regulation remains to be explored. Using stably transfected M8 cells expressing the secreted isoform (HLA-G5) of HLA-G, we investigated the role of HLA-G in inducing apoptosis and in controlling the cell cycle of activated T cells. Soluble HLA-G (HLA-G5) inhibited both CD4 and CD8 T cell proliferation. However, HLA-G5 did not induce T cell apoptosis, as determined by 3,3'-diethyloxacarbocyanine and propidium iodine labeling. It induced accumulation of the retinoblastoma protein, but not its phosphorylated and active form. Treatment of activated T cells with HLA-G5 also reduced the amounts of cyclin D2, E, A, and B by >80%. In contrast, it induced an accumulation of p27kip, but not p21cip, in activated T cells. HLA-G does not induce apoptosis of alloreactive T cells, but induces p27kip1 and inhibits cell cycle progression.     

A benzoxazine derivative specifically inhibits cell cycle progression in p53-wild type pulmonary adenocarcinoma cells

A fundamental aspect of cancer development is cancer cell proliferation. Seeking for chemical agents that can interfere with cancer cell growth has been of great interest over the years. In our study, we found that a benzoxazine derivative, (6-tert-butyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-3-yl) methanol (TBM), could inhibit cell growth and caused significant cell cycle arrest in pulmonary adenocarcinoma A549 and H460 cells with wild-type p53, while not affecting the cell cycle distribution in p53-deleted H1299 lung adenocarcinoma cells. Since P53 plays an important role in regulating cell cycle progression, we analyzed the protein level of p53 by Western blot, and detected a significant elevation of p53 level after TBM treatment in A549 and H460 cells. The data suggested that TBM might specifically inhibit the proliferation of p53 wild-type lung adenocarcinoma cells through a p53-dependent cell cycle control pathway. More interestingly, results indicated that TBM might serve as a useful tool for studying the molecular mechanisms of lung cancer cell growth and cell cycle control, especially for the biologic process regulated by P53.     

Thrombospondin-1 signaling through CD47 inhibits cell cycle progression and induces senescence in endothelial cells

CD47 signaling in endothelial cells has been shown to suppress angiogenesis, but little is known about the link between CD47 and endothelial senescence. Herein, we demonstrate that the thrombospondin-1 (TSP1)-CD47 signaling pathway is a major mechanism for driving endothelial cell senescence. CD47 deficiency in endothelial cells significantly improved their angiogenic function and attenuated their replicative senescence. Lack of CD47 also suppresses activation of cell cycle inhibitors and upregulates the expression of cell cycle promoters, leading to increased cell cycle progression. Furthermore, TSP1 significantly accelerates replicative senescence and associated cell cycle arrest in a CD47-dependent manner. These findings demonstrate that TSP1-CD47 signaling is an important mechanism driving endothelial cell senescence. Thus, TSP1 and CD47 provide attractive molecular targets for treatment of aging-associated cardiovascular dysfunction and diseases involving endothelial dysregulation.Endothelial cell (EC) senescence is accompanied with vascular dysfunction, including arterial stiffening and remodeling,¹ impaired angiogenesis,^{2, 3} reduced endothelial repair capability and increased incidence of cardiovascular disease.^{4, 5, 6} Cellular senescence can occur in vivo or in vitro in response to various stressors,^{7, 8, 9, 10} leading to suppression of cell proliferation. EC senescence has been reported to contribute to the pathogenesis of age-associated vascular diseases, such as atherosclerosis.¹¹ Thus, further understanding the mechanisms of EC senescence may help to identify effective targets for antisenescence therapy and treatment aging-associated cardiovascular disorders.Previous studies have shown that the secreted matricellular protein thrombospondin-1 (TSP1) is as potent inhibitor of angiogenesis¹² and its antiangiogenic activity is mediated by its receptors, CD36^{13, 14} and CD47.^{15, 16} CD47 is a ubiquitously expressed transmembrane protein that serves as a ligand for signal regulatory protein-α and is a signaling receptor of TSP1. The TSP1-CD47 pathway has an important role in several fundamental cellular functions, including proliferation, apoptosis, inflammation and atherosclerotic response.¹⁷ Ligation of CD47 by TSP1 has been shown to inhibit nitric oxide (NO)/cGMP signaling in vascular cells, leading to suppression of angiogenic responses.¹⁶ Recently, it was reported that lack of CD47 expression in ECs may enable these cells to spontaneously gain characteristics of embryonic stem cells.¹⁸ However, the potential role of CD47 in regulation of EC senescence has not been well explored. The present study was initiated to determine the role and mechanisms of TSP1-CD47 signaling pathway in regulating cell cycle progression and replicative senescence of ECs.     

Hydrogen peroxide inhibits cell cycle progression by inhibition of the spreading of mitotic CHO cells

Hydrogen peroxide (H(2)O(2)) induces a number of events, which are also induced by mitogens. Since the progression through the G1 phase of the cell cycle is dependent on mitogen stimulation, we were interested to study the effect of H(2)O(2) on the cell cycle progression. This study demonstrates that H(2)O(2) inhibits DNA synthesis in a dose-dependent manner when given to cells in mitosis or at different points in the G1 phase. Interestingly, mitotic cells treated immediately after synchronization are significantly more sensitive to H(2)O(2) than cells treated in the G1, and this is due to the inhibition of the cell spreading after mitosis by H(2)O(2). H(2)O(2) reversibly inhibits focal adhesion activation and stress fiber formation of mitotic cells, but not those of G1 cells. The phosphorylation of MAPK is also reversibly inhibited in both mitotic and G1 cells. Taken together, H(2)O(2) is probably responsible for the inhibition of the expression of cyclin D1 and cyclin A observed in cells in both phases. In conclusion, H(2)O(2) inhibits cell cycle progression by inhibition of the spreading of mitotic CHO cells. This may play a role in pathological processes in which H(2)O(2) is generated.     

Acetylcholine inhibits cell cycle progression in rat Schwann cells by activation of the M2 receptor subtype

Cultures of Schwann cells from neonatal rat sciatic nerves were treated with acetylcholine agonists and the effects on cell proliferation evaluated. (3)[H]-thymidine incorporation shows that acetylcholine (ACh) receptor agonists inhibit cell proliferation, and FACS analysis demonstrates cell-cycle arrest and accumulation of cells in the G1 phase. The use of arecaidine, a selective agonist of muscarinic M2 receptors reveals that this effect depends mainly on M2 receptor activation. The arecaidine dependent-block in G1 is reversible because removal of arecaidine from the culture medium induces progression to the S phase. The block of the G1-S transition is also characterized by modulation of the expression of several cell-cycle markers. Moreover, treatment with ACh receptor agonist causes both a decrease in the PCNA protein levels in Schwann cell nuclei and an increase in p27 and p53 proteins. Finally, immuno-electron microscopy demonstrates that M2 receptors are expressed by Schwann cells in vivo. These results indicate that ACh, by modulating Schwann cell proliferation through M2 receptor activation, might contribute to their progression to a more differentiated phenotype.     

RPS27a promotes proliferation,regulates cell cycle progression and inhibits apoptosis of leukemia cells

Ribosomal protein S27a (RPS27a) could perform extra-ribosomal functions besides imparting a role in ribosome biogenesis and post-translational modifications of proteins. The high expression level of RPS27a was reported in solid tumors, and we found that the expression level of RPS27a was up-regulated in advanced-phase chronic myeloid leukemia (CML) and acute leukemia (AL) patients. In this study, we explored the function of RPS27a in leukemia cells by using CML cell line K562 cells and its imatinib resistant cell line K562/G01 cells. It was observed that the expression level of RPS27a was high in K562 cells and even higher in K562/G01 cells. Further analysis revealed that RPS27a knockdown by shRNA in both K562 and K562G01 cells inhibited the cell viability, induced cell cycle arrest at S and G2/M phases and increased cell apoptosis induced by imatinib. Combination of shRNA with imatinib treatment could lead to more cleaved PARP and cleaved caspase-3 expression in RPS27a knockdown cells. Further, it was found that phospho-ERK(p-ERK) and BCL-2 were down-regulated and P21 up-regulated in RPS27a knockdown cells. In conclusion, RPS27a promotes proliferation, regulates cell cycle progression and inhibits apoptosis of leukemia cells. It appears that drugs targeting RPS27a combining with tyrosine kinase inhibitor (TKI) might represent a novel therapy strategy in TKI resistant CML patients.