Heme oxygenase-1 expression levels are cell cycle dependent

Heme oxygenase-1 (HO-1) is a stress protein, which has been suggested to participate in defense mechanisms against agents that may induce oxidative injury, such as angiotensin II (Ang II). The purpose of the present study was to examine the role of human HO-1 in cell-cycle progression. We investigated the effect of Ang II on HO-1 gene expression in serum-deprived media to drive human endothelial cells into G(0)/G(1) (1% FBS) compared to exponentially grown cells (10% FBS). The addition of Ang II (100 ng/ml) to endothelial cells increased HO-1 protein and activity in G(0)/G(1) in a time-dependent manner, reaching a maximum HO-1 level at 16 h. Real-time RT-PCR demonstrated that Ang II increased the levels of HO-1 mRNA in G(0)/G(1) as early as 1 h. The rate of HO-1 induction in response to Ang II was several-fold higher in serum-starved cells compared to cells cultured in continuous 10% FBS. The addition of Ang II increased the generation of 8-epi-isoprostane PGF(2 alpha). Inhibition of HO-1, by Stannis mesoporphyrin (SnMP), potentiated Ang II-mediated DNA damage and generation of 8-epi-isoprostane PGF(2 alpha). These results imply that expression of HO-1 in G(0)/G(1), in the presence of Ang II, may be a key player in attenuating DNA damage during cell-cycle progression. Thus, exposure of endothelial cells to Ang II causes a complex response involving generation of superoxide anion, which may be involved in DNA damage. Upregulation of HO-1 ensures the generation of bilirubin and carbon monoxide (CO) in G(0)/G(1) phase to counteract Ang II-mediated oxidative DNA damage. Inducibility of HO-1 in G(0)/G(1) phase is essential and probably regulated by a complex system involving oxygen species to assure controlled cell growth.     

8-Hydroxydeoxyguanosine induces senescence-like changes in KG-1, human acute myelocytic leukemia cell line

8-Hydroxydeoxyguanosine (oh⁸dG) treatment induced senescence-like changes in KG-1 cells, a human acute myelocytic leukemia cell line. The oh⁸dG-treated cells stained positive for senescence associated β-galactosidase (SA-β-galactosidase) and had enlarged cell shape, both of which are senescence indexes. The oh⁸dG-treated cells were also cell growth inhibited and arrested at G₁ in the cell cycle. The accumulation of cdk (cyclin dependent kinase) inhibitors, such as p16, p21, and p27, also implies that cellular senescence was induced in oh⁸dG-treated cells. However, these changes were not accompanied by cell differentiation or telomerase activity. Taken together, we conclude that oh⁸dG treatment of KG-1 cells induces cellular senescence.     

Prolonged prostaglandin E1 stimulation of cyclic AMP production in transformed and normal WI-38 fibroblasts

Summary Long-term (48-hr) incubations of either the fibroblast strain WI-48 or its SV40-transformed counterpart, WI-38-VA13-2RA, in growth medium containing 1 μm prostaglandin E₁ (PGE₁) resulted in a sustained production and release of cyclic AMP from the cells into the medium. Despite the steady production, intracellular levels of the nucleotide decreased, reaching steady-state values within 4 hr of the initial exposure to PGE₁. These values were maintained for the remainder of the 48-hr experimental period. The steadystate levels of intracellular cyclic AMP were higher than those observed in unstimulated cells, and cyclic AMP-dependent protein phosphokinase was in a highly activated state as compared to controls. Under these conditions little change in the growth or morphology of either the normal or transformed cells was observed. In contrast, inhibition of growth, apparent cell death, and unusual morphological changes were observed in both normal and transformed cells when high concentrations of either PGE₁ (10 μm) or the phosphodiesterase inhibitor 1-methyl, 3-isobutylxanthine (0.5mm to 2mm) were used, which was indicative of toxic effects of the drugs. It was concluded that cyclic AMP-mediated activation of protein phosphokinase does not completely inhibit growth in WI-38 cells or restore normal growth and morphology to the SV40-transformed cells. This work was supported by Grants AM 13904 and CA 21612 from the National Institutes of Health, Department of Health, Education and Welfare.     

Inhibition of PI3K by ZSTK474 suppressed tumor growth not via apoptosis but G0/G1 arrest

Phosphoinositide 3-kinase (PI3K) is a potential target in cancer therapy. Inhibition of PI3K is believed to induce apoptosis. We recently developed a novel PI3K inhibitor ZSTK474 with antitumor efficacy. In this study, we have examined the underlying mode of action by which ZSTK474 exerts its antitumor efficacy. In vivo, ZSTK474 effectively inhibited the growth of human cancer xenografts. In parallel, ZSTK474 treatment suppressed the expression of phospho-Akt, suggesting effective PI3K inhibition, and also suppressed the expression of nuclear cyclin D1 and Ki67, both of which are hallmarks of proliferation. However, ZSTK474 treatment did not increase TUNEL-positive apoptotic cells. In vitro, ZSTK474 induced marked G₀/G₁ arrest, but did not increase the subdiploid cells or activate caspase, both of which are hallmarks of apoptosis. These results clearly indicated that inhibition of PI3K by ZSTK474 did not induce apoptosis but rather induced strong G₀/G₁ arrest, which might cause its efficacy in tumor cells.     

BC-N102 suppress breast cancer tumorigenesis by interfering with cell cycle regulatory proteins and hormonal signaling,and induction of time-course arrest of cell cycle at G1/G0 phase

Mechanisms of breast cancer progression and invasion, often involve alteration of hormonal signaling, and upregulation and/or activation of signal transduction pathways that input to cell cycle regulation. Herein, we describe a rationally designed first-in-class novel small molecule inhibitor for targeting oncogenic and hormonal signaling in ER-positive breast cancer. BC-N102 treatment exhibits dose-dependent cytotoxic effects against ER+ breast cancer cell lines. BC-N102 exhibited time course- and dose-dependent cell cycle arrest via downregulation of the estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), phosphatidylinositol 3-kinase (PI3K), phosphorylated (p)-extracellular signal-regulated kinase (ERK), p-Akt, CDK2, and CDK4 while increasing p38 mitogen-activated protein kinase (MAPK), and mineralocorticoid receptor (MR) signaling in breast cancer cell line. In addition, we found that BC-N102 suppressed breast cancer tumorigenesis in vivo and prolonged the survival of animals. Our results suggest that the proper application of BC-N102 may be a beneficial chemotherapeutic strategy for ER+ breast cancer patients.     

Prolyl oligopeptidase participates in cell cycle progression in a human neuroblastoma cell line

Prolyl oligopeptidase (POP) is a post-proline cleaving enzyme, which is widely distributed in various organs, with high levels in the brain. In this study, we investigated the effects of a selective POP inhibitor, 3-({4-[2-(E)-styrylphenoxy]butanoyl}-l-4-hydroxyprolyl)-thiazolidine (SUAM-14746), on the growth of NB-1 human neuroblastoma cells. SUAM-14746 treatment for 24–72 h suppresses the growth of NB-1 cells without cell death in a dose-dependent manner (10–60 μM). Similar suppressive effects were observed with another POP inhibitor benzyloxycarbonyl-thioprolyl-thioprolinal. The SUAM-14746-induced growth inhibition in NB-1 cells was associated with pronounced G₀/G₁ arrest and reduced levels of phosphorylated retinoblastoma protein (pRb), cyclin E, and cyclin dependent kinase (CDK) 2, and increased levels of the CDK inhibitor p27^kip1 and the tumor suppressor p53. SUAM-14746 also induced transient inhibition of S and G₂/M phase progression, which was correlated with retardation of the decrease in the levels of cyclins A and B. Moreover, RNAi-mediated knockdown of POP also led to inhibition of NB-1 cell growth and the effect was accompanied by G₀/G₁ arrest. These results indicate that POP is a part of the machinery that controls the cell cycle.     

The Telomere-Binding Protein Taz1p as a Target for Modification by a SUMO-1 Homologue in Fission Yeast

In fission yeast (Schizosaccharomyces pombe) the homologue of the mammalian SUMO-1 ubiquitin-like modifier is encoded by the pmt3 gene. A two-hybrid screen using the telomere-binding protein Taz1p as bait identified Pmt3p as an interacting factor. In vitro experiments using purified components of the fission yeast Pmt3p modification system demonstrated that Taz1p could be modified directly by Pmt3p. The amino acid sequence of Taz1p contains a close match to the consensus modification site for SUMO-1, and a PEST sequence similar to those found in established SUMO-1 targets. Although previous experiments have identified an increase in telomere length as one consequence of the pmt3– genotype, we could not detect Pmt3p modification of Taz1p in protein extracts made from exponentially growing haploid cells or any effect of Pmt3p on the localization of GFP-Taz1p at discrete foci in the haploid cell nucleus.     

Overexpression of the Inhibitor Protein IF1 in AS-30D Hepatoma Produces a Higher Association with Mitochondrial F1F0 ATP Synthase Compared to Normal Rat Liver: Functional and Cross-Linking Studies

According to functional studies, the higher IF(1) content reported in mitochondria of cancerous cells is supposed to induce a higher association with the F(1)F(0) complex than in normal cells and therefore a better inhibition of its ATPase activity. The first structural evidence supporting this prediction is here presented. Densitometric analyses of Western blotting experiments indicated a 2-fold increase in IF(1) content of AS-30D submitochondrial particles compared to normal rat liver controls. The ratio of IF(1)/F(1) alpha subunit increased similarly as judged by Westernblot analyses. This IF(1) overexpression correlated with a slower rate of IF(1) release (F(1)F(0)-ATPase activation) from the F(1)F(0) complex in AS-30D than in normal rat liver submitochondrial particles. The IF(1)-IF(1), gamma-IF(1), and alpha-IF(1) cross-linkages previously formed with dithiobis(succinimidylpropionate) in bovine F(1)F(0)I and IF(1) complexes were reproduced in the F(1)F(0)I-ATP synthase of hepatoma AS-30D cells. However, a much lower yield of IF(1) cross-linkages was found in normal rat liver particles which made them almost undetectable in SMP as well as in the immunoprecipitated F(1)F(0)I complex. Modeling in vivo IF(1) overexpression of cancerous cells by in vitro reconstitution of excess recombinant IF(1) with rat liver submitochondrial particles devoid of IF(1) reproduced the same IF(1) cross-linkages observed in AS-30D particles.     

Phorbol ester potentiates the growth inhibitory effects of troglitazone via up-regulation of PPARgamma in A549 cells

The activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to induce growth arrest and differentiation of various cancer cells. In the current study, we investigated the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of PPARgamma and proliferation of A549 cells. TPA elicited a dose- and time-dependent increase in PPARgamma mRNA and protein levels. PPARgamma expression in response to TPA was attenuated by pretreatment with bisindolylmaleimide I, N-acetyl-L-cysteine (NAC) and PD98059. TPA-induced protein kinase C (PKC) activation was linked to the generation of reactive oxygen species (ROS), both of which were indispensable for PPARgamma expression in A549 cells. Pretreatment with bisindolylmaleimide I or NAC blocked TPA-induced phosphorylation of extracellular signal-regulated kinase (ERK), suggesting that ERK-mediated signaling is also involved in the induction of PPARgamma. Furthermore, the growth inhibitory effect of troglitazone was significantly potentiated by prolonged incubation with TPA and was attenuated in the presence of GW9662, a specific inhibitor of PPARgamma. These effects were associated with an induction of cell cycle arrest at G0/G1 phase, which was accompanied by the induction of p21Waf1/Cip1 expression and decreased cyclin D1 expression. Taken together, these observations indicate that TPA synergizes with PPARgamma ligand to inhibit cell growth through up-regulation of PPARgamma expression.     

Antitumor effects of a novel benzonaphthofurandione derivative (8e) on the human colon cancer cells in vitro and in vivo through cell cycle arrest accompanied with the modulation of EGFR and mTOR signaling

Benzonaphthofurandione has been considered as an important class of naturally occurring and synthetic compounds having a variety of biological functions. In this study, we evaluated the antitumor effects of 3-[2-(dimethylamino)isopropoxy]-1-hydroxybenzo[b]naphtho[2,3-d]furan-6,11-dione (8e), a novel benzonaphthofurandione derivative, on the growth of colorectal cancer HCT 116 cells both in vitro culture and an in vivo animal model.Compound 8e exhibited the potential growth inhibition of the colon cancer cells in a concentration-dependent manner. The anti-proliferative activity of 8e was also associated with the induction of cell cycle arrest in the G₀/G₁ phase. The 8e-induced cell cycle arrest was well correlated with the suppression of cyclin-dependent kinase 2 (CDK2), CDK4, cyclin D1, cyclin E, c-Myc, and phosphorylated retinoblastoma protein (pRb). The tumor growth in xenograft nude mice bearing HCT 116 cells by compound 8e (10 mg/kg) also significantly inhibited without any overt toxicity. In addition, the down-regulation of epidermal growth factor receptor (EGFR), Akt, and mTOR signalings were associated with the anti-proliferative activity of compound 8e in colon cancer cells. Taken together, these findings suggested that cell cycle arrest and modulation of cell signal transduction pathways might be the plausible mechanisms of actions for the anti-proliferative activity of 8e, and thus 8e might be used as an effective chemotherapeutic agent in human colon cancer.     

Houttuynia cordata Thunb extract modulates G0/G1 arrest and Fas/CD95-mediated death receptor apoptotic cell death in human lung cancer A549 cells

Background

Houttuynia cordata Thunb (HCT) is commonly used in Taiwan and other Asian countries as an anti-inflammatory, antibacterial and antiviral herbal medicine. In this study, we investigated the anti-human lung cancer activity and growth inhibition mechanisms of HCT in human lung cancer A549 cells.

Results

In order to investigate effects of HCT on A549 cells, MTT assay was used to evaluate cell viability. Flow cytometry was employed for cell cycle analysis, DAPI staining, and the Comet assay was used for DNA fragmentation and DNA condensation. Western blot analysis was used to analyze cell cycle and apoptotic related protein levels. HCT induced morphological changes including cell shrinkage and rounding. HCT increased the G₀/G₁ and Sub-G₁ cell (apoptosis) populations and HCT increased DNA fragmentation and DNA condensation as revealed by DAPI staining and the Comet assay. HCT induced activation of caspase-8 and caspase-3. Fas/CD95 protein levels were increased in HCT-treated A549 cells. The G₀/G₁ phase and apoptotic related protein levels of cyclin D1, cyclin A, CDK 4 and CDK 2 were decreased, and p27, caspase-8 and caspase-3 were increased in A549 cells after HCT treatment.

Conclusions

The results demonstrated that HCT-induced G₀/G₁ phase arrest and Fas/CD95-dependent apoptotic cell death in A549 cells     

The Inhibitor Protein (IF1) of the F1F0-ATPase Modulates Human Osteosarcoma Cell Bioenergetics

The bioenergetics of IF₁ transiently silenced cancer cells has been extensively investigated, but the role of IF₁ (the natural inhibitor protein of F₁F₀-ATPase) in cancer cell metabolism is still uncertain. To shed light on this issue, we established a method to prepare stably IF₁-silenced human osteosarcoma clones and explored the bioenergetics of IF₁ null cancer cells. We showed that IF₁-silenced cells proliferate normally, consume glucose, and release lactate as controls do, and contain a normal steady-state ATP level. However, IF₁-silenced cells displayed an enhanced steady-state mitochondrial membrane potential and consistently showed a reduced ADP-stimulated respiration rate. In the parental cells (i.e. control cells containing IF₁) the inhibitor protein was found to be associated with the dimeric form of the ATP synthase complex, therefore we propose that the interaction of IF₁ with the complex either directly, by increasing the catalytic activity of the enzyme, or indirectly, by improving the structure of mitochondrial cristae, can increase the oxidative phosphorylation rate in osteosarcoma cells grown under normoxic conditions.     

Modulation at a Distance of Proton Conductance through the Saccharomyces cerevisiae Mitochondrial F1F0-ATP Synthase by Variants of the Oligomycin Sensitivity-Conferring Protein Containing Substitutions near the C-Terminus

We have sought to elucidate how the oligomycin sensitivity-conferring protein (OSCP) of the mitochondrial F₁F₀-ATP synthase (mtATPase) can influence proton channel function. Variants of OSCP, from the yeast Saccharomyces cerevisiae, having amino acid substitutions at a strictly conserved residue (Gly166) were expressed in place of normal OSCP. Cells expressing the OSCP variants were able to grow on nonfermentable substrates, albeit with some increase in generation time. Moreover, these strains exhibited increased sensitivity to oligomycin, suggestive of modification in functional interactions between the F₁ and F₀ sectors mediated by OSCP. Bioenergetic analysis of mitochondria from cells expressing OSCP variants indicated an increased respiratory rate under conditions of no net ATP synthesis. Using specific inhibitors of mtATPase, in conjunction with measurement of changes in mitochondrial transmembrane potential, it was revealed that this increased respiratory rate was a result of increased proton flux through the F₀ sector. This proton conductance, which is not coupled to phosphorylation, is exquisitely sensitive to inhibition by oligomycin. Nevertheless, the oxidative phosphorylation capacity of these mitochondria from cells expressing OSCP variants was no different to that of the control. These results suggest that the incorporation of OSCP variants into functional ATP synthase complexes can display effects in the control of proton flux through the F₀ sector, most likely mediated through altered protein—protein contacts within the enzyme complex. This conclusion is supported by data indicating impaired stability of solubilized mtATPase complexes that is not, however, reflected in the assembly of functional enzyme complexes in vivo. Given a location for OSCP atop the F₁-₃₃ hexamer that is distant from the proton channel, then the modulation of proton flux by OSCP must occur at a distance. We consider how subtle conformational changes in OSCP may be transmitted to F₀.     

Molecular mechanisms of nitric oxide-induced growth arrest and apoptosis in fetal pulmonary arterial smooth muscle cells

Superoxide plays an important role in pulmonary arterial smooth muscle cell (SMC) proliferation and survival. The rapid reaction between superoxide and nitric oxide (NO) to form peroxynitrite suggests that endothelium-derived NO may influence adjacent SMC growth. To investigate this possibility, we determined the dose-dependent effects of NO on the proliferation and viability of pulmonary arterial SMC isolated from fetal lambs (FPASMC). Using fluorescence microscopy we found a dose-dependent decrease in superoxide levels in FPASMC treated with the NO donor spermine NONOate. This was associated with an increase in peroxynitrite-mediated protein nitration. At doses between 50 and 250 microM, spermine NONOate attenuated serum-induced FPASMC proliferation resulting in a G(0)/G(1) cell cycle arrest. This process involved a decrease in levels of cyclin A and an increase in the nuclear localization of p21 and p27. Furthermore, 500 microM spermine NONOate decreased viable cell number by inducing programmed cell death: FPASMC treated with 500 microM spermine NONOate displayed a loss of mitochondrial membrane potential, followed by caspase activation and DNA fragmentation. These data suggest that NO inhibits superoxide-induced proliferation of FPASMC and at higher doses induces apoptosis. NO donors may therefore prove to be useful therapeutic tools to treat diseases resulting from excessive proliferation of vascular smooth muscle.     

Inhibition of cancer cell growth by cyclin dependent kinase 4 inhibitors synthesized based on the structure of fascaplysin

Tryptamine derivatives, a new structural class of cyclin dependent kinase 4 inhibitors, have been identified during extensive biological screening of synthetic molecules. The molecules were synthesized based on the structure of fascaplysin, which is not only a specific inhibitor of the Cdk4-cyclin D1 enzyme but also a relatively toxic molecule, probably because it binds and intercalates DNA. Interestingly, the new structural analogues of fascaplysin do not interact or intercalate with double-stranded DNA, although they inhibit Cdk4-cyclin D1 specifically. We found that compound CA199 was the most potent molecule, showing at least 25-fold specificity towards Cdk4-cyclin D1 (IC50 for Cdk4-cyclin D1 = 20 microM, Cdk2 > 500 microM). CA199 inhibits the growth of different cancer cell lines at concentrations ranging from 10-40 microM. It blocks growth of asynchronous cells at G0/G1 in a retinoblastoma protein (pRb) dependent manner. Moreover, CA199 blocks growth only at early G1 in synchronised cells released from a mimosine-induced G1/S block. These observations are reminiscent of a true Cdk4 inhibitor.     

Apoptosis signal-regulating kinase 1 is an intracellular inducer of p38 MAPK-mediated myogenic signalling in cardiac myoblasts

Myogenic differentiation is an essential process for the myogenesis in response to various extracellular stimuli. p38 MAPK is a core signalling molecule in myogenic differentiation. The activation of p38 MAPK is required for myogenic differentiation; however, the mechanism for this activation remains undefined. ASK1 is a member of the MAP3K family that activates both JNK and p38 MAPK pathways in response to an array of stresses such as oxidative stress, endoplasmic reticulum stress and calcium influx. Here, we reported that TNFα was significantly released from H9c2 cardiac myoblast in differentiation medium. Furthermore, the oxidant H₂O₂ acted as a messenger in the TNFα signalling pathway to disrupt the complex of ASK1-Trx, which was followed by the activation of ASK1 in cardiac myogenic differentiation. Subsequently, the activated ASK1 stimulated MKK3/6-p38MAPK signalling cascade to induce specific myogenic differentiation. In addition, exogenous TNFα added to the medium at physiological levels enhanced the ASK1-p38 MAPK signalling pathway through the increased generation of H₂O₂. Interestingly, inhibition of p38 MAPK abrogated the production of H₂O₂, suggesting that there might be a positive feedback loop in the myogenic-redox signalling pathway. These results indicate that ASK1 is a new intracellular regulator of activation of the p38 MAPK in cardiac myogenic differentiation.     

Sphingosine-1-phosphate receptor S1P1 is regulated by direct interactions with P-Rex1, a Rac guanine nucleotide exchange factor

Sphingosine-1-phosphate (S1P) receptors S1P₁ are emerging molecular targets for the treatment of cancer, vascular and immune diseases, due to their pivotal role in cell migration and survival of immune and endothelial cells. A therapeutic strategy to control S1P₁ function is based on agonists that promote changes on S1P₁ expression at the plasma membrane. Here, we explored the hypothesis that cell surface expression and function of S1P₁ are influenced by direct interactions with P-Rex1, a guanine nucleotide exchange factor for Rac. We demonstrate that P-Rex1-PDZ domains interact with S1P₁-carboxyl terminal tail and full length receptor monomers and dimers. Endothelial cells transfected with P-Rex1-PDZ domains show an increased migratory response to S1P. S1P₁ trafficking to intracellular compartments is diminished by coexpression of P-Rex1. We conclude that S1P₁ signaling linked to cell migration is facilitated by a functional interaction with P-Rex1 via a mechanism that involves the maintenance of S1P₁ receptors at the cell membrane.     

Post-opioid receptor adaptations to chronic morphine; altered functionality and associations of signaling molecules

Opioid desensitization/tolerance mechanisms have largely focused on adaptations that occur on the level of the mu-opioid receptor (MOR) itself. These include opioid receptor phosphorylation and ensuing trafficking events. Recent research, however, has revealed additional adaptations that occur downstream from the opioid receptor, which involve covalent modification of signaling molecules and altered associations among them. These include augmented isoform-specific synthesis of adenylyl cyclase (AC) and their phosphorylation as well as augmented phosphorylation of the G(beta) subunit of G(beta gamma). The aggregate effect of these changes is to shift mu-opioid receptor-coupled signaling from predominantly G(i alpha) inhibitory to (G(i)-derived) G(beta gamma) stimulatory AC signaling. Most recently, chronic morphine has been shown to enhance the association (interaction) between MOR and G(s), which should provide an additional avenue for offsetting inhibitory MOR signaling sequelae. The unfolding complexity of chronic morphine-induced sequelae demands an evolving broader and more encompassing perspective on opioid tolerance-producing mechanisms. This should facilitate understanding tolerance within the context of physiological plasticity that is activated by chronic exposure to drugs of abuse. Additional research is required to integrate the various tolerance-producing adaptations that have been elucidated to date. Specifically, the relative contribution to opioid tolerance of identified adaptations is still unknown as is the extent to which they vary among different regions of the central nervous system.