Reevesioside A,a Cardenolide Glycoside,Induces Anticancer Activity against Human Hormone-Refractory Prostate Cancers through Suppression of c-myc Expression and Induction of G1 Arrest of the Cell Cycle

In the past decade, there has been a profound increase in the number of studies revealing that cardenolide glycosides display inhibitory activity on the growth of human cancer cells. The use of potential cardenolide glycosides may be a worthwhile approach in anticancer research. Reevesioside A, a cardenolide glycoside isolated from the root of Reevesia formosana, displayed potent anti-proliferative activity against human hormone-refractory prostate cancers. A good correlation (r² = 0.98) between the expression of Na⁺/K⁺-ATPase α₃ subunit and anti-proliferative activity suggested the critical role of the α₃ subunit. Reevesioside A induced G1 arrest of the cell cycle and subsequent apoptosis in a thymidine block-mediated synchronization model. The data were supported by the down-regulation of several related cell cycle regulators, including cyclin D1, cyclin E and CDC25A. Reevesioside A also caused a profound decrease of RB phosphorylation, leading to an increased association between RB and E2F1 and the subsequent suppression of E2F1 activity. The protein and mRNA levels of c-myc, which can activate expression of many downstream cell cycle regulators, were dramatically inhibited by reevesioside A. Transient transfection of c-myc inhibited the down-regulation of both cyclin D1 and cyclin E protein expression to reevesioside A action, suggesting that c-myc functioned as an upstream regulator. Flow cytometric analysis of JC-1 staining demonstrated that reevesioside A also induced the significant loss of mitochondrial membrane potential. In summary, the data suggest that reevesioside A inhibits c-myc expression and down-regulates the expression of CDC25A, cyclin D1 and cyclin E, leading to a profound decrease of RB phosphorylation. G1 arrest is, therefore, induced through E2F1 suppression. Consequently, reevesioside A causes mitochondrial damage and an ultimate apoptosis in human hormone-refractory prostate cancer cells.     

Cytosolic phospholipase A2α sustains pAKT,pERK and AR levels in PTEN-null/mutated prostate cancer cells

Constitutive phosphorylation of protein kinase B (AKT) is a common feature of cancer caused by genetic alteration in the phosphatase and tensin homolog (PTEN) gene and is associated with poor prognosis. This study determined the role of cytosolic phospholipase A₂α (cPLA₂α) in AKT, extracellular signal-regulated kinase (ERK) and androgen receptor (AR) signaling in PTEN-null/mutated prostate cancer cells. Doxycycline (Dox)-induced expression of cPLA₂α led to an increase in pAKT, pGSK3β and cyclin D1 levels in LNCaP cells that possess a PTEN frame-shift mutation. In contrast, silencing cPLA₂α expression with siRNA decreased pAKT, pGSK3β and cyclin D1 levels in both PC-3 (PTEN deletion) and LNCaP cells. Silencing of cPLA₂α decreased pERK and AR protein levels. The inhibitory effect of cPLA₂α siRNA on pAKT and AR protein levels was reduced by the addition of arachidonic acid (AA), whereas the stimulatory effect of AA on pAKT, pERK and AR levels was decreased by an inhibitor of 5-hydroxyeicosatetraenoic acid production. Pharmacological blockade of cPLA₂α with Efipladib reduced pAKT and AR levels with a concomitant inhibition of PC-3 and LNCaP cell proliferation. These results demonstrate an important role for cPLA₂α in sustaining AKT, ERK and AR signaling in PTEN-null/mutated prostate cancer cells and provide a potential molecular target for treating prostate cancer.     

Regulation of Exit from Quiescence by p27 and Cyclin D1-CDK4

The synthesis of cyclin D1 and its assembly with cyclin-dependent kinase 4 (CDK4) to form an active complex is a rate-limiting step in progression through the G₁ phase of the cell cycle. Using an activated allele of mitogen-activated protein kinase kinase 1 (MEK1), we show that this kinase plays a significant role in positively regulating the expression of cyclin D1. This was found both in quiescent serum-starved cells and in cells expressing dominant-negative Ras. Despite the observation that cyclin D1 is a target of MEK1, in cycling cells, activated MEK1, but not cyclin D1, is capable of overcoming a G₁ arrest induced by Ras inactivation. Either wild-type or catalytically inactive CDK4 cooperates with cyclin D1 in reversing the G₁ arrest induced by inhibition of Ras activity. In quiescent NIH 3T3 cells expressing either ectopic cyclin D1 or activated MEK1, cyclin D1 is able to efficiently associate with CDK4; however, the complex is inactive. A significant percentage of the cyclin D1-CDK4 complexes are associated with p27 in serum-starved activated MEK1 or cyclin D1 cell lines. Reduction of p27 levels by expression of antisense p27 allows for S-phase entry from quiescence in NIH 3T3 cells expressing ectopic cyclin D1, but not in parental cells.     

Liganded Thyroid Hormone Receptor-α Enhances Proliferation of Pancreatic β-Cells

Failure of the functional pancreatic β-cell mass to expand in response to increased metabolic demand is a hallmark of type 2 diabetes. Lineage tracing studies indicate that replication of existing β-cells is important for β-cell proliferation in adult animals. In rat pancreatic β-cell lines (RIN5F), treatment with 100 nm thyroid hormone (triiodothyronine, T₃) enhances cell proliferation. This result suggests that T₃ is required for β-cell proliferation or replication. To identify the role of thyroid hormone receptor α (TRα) in the processes of β-cell growth and cell cycle regulation, we constructed a recombinant adenovirus vector, AdTRα. Infection with AdTRα to RIN5F cells increased the expression of cyclin D1 mRNA and protein. Overexpression of the cyclin D1 protein in AdTRα-infected cells led to activation of the cyclin D1/cyclin-dependent kinase/retinoblastoma protein/E2F pathway, along with cell cycle progression and cell proliferation following treatment with 100 nm T₃. Conversely, lowering cellular cyclin D1 by small interfering RNA knockdown in AdTRα-infected cells led to down-regulation of the cyclin D1/CDK/Rb/E2F pathway and inhibited cell proliferation. Furthermore, in immunodeficient mice with streptozotocin-induced diabetes, intrapancreatic injection of AdTRα led to the restoration of islet function and to an increase in the β-cell mass. These results support the hypothesis that liganded TRα plays a critical role in β-cell replication and in expansion of the β-cell mass during postnatal development. Thus, liganded TRα may be a target for therapeutic strategies that can induce the expansion and regeneration of β-cells.     

Mechanism of the cytotoxicity of the diazoparaquinone antitumor antibiotic kinamycin F

The bacterial metabolite kinamycin F, which is being investigated as a potent antitumor agent, contains an unusual and potentially reactive diazo group, a paraquinone, and a phenol functional group. Kinamycin F reacted with glutathione (GSH) in a complex series of reactions which suggested that kinamycin F may have its cytotoxicity modulated by GSH. Consistent with this idea, 2-oxo-4-thiazolidinecarboxylic acid treatment to increase cellular GSH levels and buthionine sulfoximine treatment to decrease GSH levels resulted in decreased and increased kinamycin F cytotoxicity, respectively, in K562 leukemia cells. Kinamycin F weakly bound to DNA and induced DNA damage in K562 cells that was independent of GSH levels. The GSH-promoted DNA nicking induced by kinamycin F in vitro was attenuated by deferoxamine, dimethyl sulfoxide, and catalase, which indicated that DNA damage initiated by this agent occurred in an iron-, hydrogen-peroxide-, and hydroxyl-radical-dependent manner. Electron paramagnetic resonance spectroscopy experiments showed that the GSH/kinamycin F system produced a semiquinone free radical and that the hydrogen peroxide/peroxidase/kinamycin F system generated a phenoxyl free radical. In conclusion, the results indicated that kinamycin F cytotoxicity may be due to reductive and/or peroxidative activation to produce DNA-and protein-damaging species.     

PACSIN 2 represses cellular migration through direct association with cyclin D1 but not its alternate splice form cyclin D1b

Cyclin D1 overexpression is a common feature of many human malignancies. Genomic deletion analysis has demonstrated a key role for cyclin D1 in cellular proliferation, angiogenesis and cellular migration. To investigate the mechanisms contributing to cyclin D1 functions, we purified cyclin D1a-associated complexes by affinity chromatography and identified the PACSIN 2 (protein kinase C and casein kinase substrate in neurons 2) protein by mass spectrometry. The PACSIN 2, but not the related PACSIN 1 and 3, directly bound wild-type cyclin D1 (cyclin D1a) at the carboxyl terminus and failed to bind cyclin D1b, the alternative splicing variant of cyclin D1. PACSIN 2 knockdown induced cellular migration and reduced cell spreading in LNCaP cells expressing cyclin D1a. In cyclin D1^−/− mouse embryonic fibroblasts (MEFs), cyclin D1a, but not cyclin D1b, reduced the cell spreading to a polarized morphology. siPACSIN 2 had no effect on cellular migration of cyclin D1^−/− MEFs. Cyclin D1a restored the migratory ability of cyclin D1^−/− MEFs, which was further enhanced by knocking down PACSIN 2 with siRNA. The cyclin D1-associated protein, PACSIN 2, regulates cell spreading and migration, which are dependent on cyclin D1 expression.Key words: PACSIN 2, cyclin D1, polymorphism, cellular migration, cell spreading, cancer     

Nucleolar GTP-binding Protein-1 (NGP-1) Promotes G1 to S Phase Transition by Activating Cyclin-dependent Kinase Inhibitor p21Cip1/Waf1

Nucleolar GTP-binding protein (NGP-1) is overexpressed in various cancers and proliferating cells, but the functional significance remains unknown. In this study, we show that NGP-1 promotes G₁ to S phase transition of cells by enhancing CDK inhibitor p21^Cip-1/Waf1 expression through p53. In addition, our results suggest that activation of the cyclin D1-CDK4 complex by NGP-1 via maintaining the stoichiometry between cyclin D1-CDK4 complex and p21 resulted in hyperphosphorylation of retinoblastoma protein at serine 780 (p-RB^Ser-780) followed by the up-regulation of E2F1 target genes required to promote G₁ to S phase transition. Furthermore, our data suggest that ribosomal protein RPL23A interacts with NGP-1 and abolishes NGP-1-induced p53 activity by enhancing Mdm2-mediated p53 polyubiquitination. Finally, reduction of p-RB^Ser-780 levels and E2F1 target gene expression upon ectopic expression of RPL23a resulted in arrest at the G₁ phase of the cell cycle. Collectively, this investigation provides evidence that NGP-1 promotes cell cycle progression through the activation of the p53/p21^Cip-1/Waf1 pathway.     

Regulation of glioblastoma progression by cord blood stem cells is mediated by downregulation of cyclin D1

Background

The normal progression of the cell cycle requires sequential expression of cyclins. Rapid induction of cyclin D1 and its associated binding with cyclin-dependent kinases, in the presence or absence of mitogenic signals, often is considered a rate-limiting step during cell cycle progression through the G₁ phase.

Methodology/Principal Findings

In the present study, human umbilical cord blood stem cells (hUCBSC) in co-cultures with glioblastoma cells (U251 and 5310) not only induced G₀-G₁ phase arrest, but also reduced the number of cells at S and G₂-M phases of cell cycle. Cell cycle regulatory proteins showed decreased expression levels upon treatment with hUCBSC as revealed by Western and FACS analyses. Inhibition of cyclin D1 activity by hUCBSC treatment is sufficient to abolish the expression levels of Cdk 4, Cdk 6, cyclin B1, β-Catenin levels. Our immuno precipitation experiments present evidence that, treatment of glioma cells with hUCBSC leads to the arrest of cell-cycle progression through inactivation of both cyclin D1/Cdk 4 and cyclin D1/Cdk 6 complexes. It is observed that hUCBSC, when co-cultured with glioma cells, caused an increased G₀-G₁ phase despite the reduction of G₀-G₁ regulatory proteins cyclin D1 and Cdk 4. We found that this reduction of G₀-G₁ regulatory proteins, cyclin D1 and Cdk 4 may be in part compensated by the expression of cyclin E1, when co-cultured with hUCBSC. Co-localization experiments under in vivo conditions in nude mice brain xenografts with cyclin D1 and CD81 antibodies demonstrated, decreased expression of cyclin D1 in the presence of hUCBSC.

Conclusions/Significance

This paper elucidates a model to regulate glioma cell cycle progression in which hUCBSC acts to control cyclin D1 induction and in concert its partner kinases, Cdk 4 and Cdk 6 by mediating cell cycle arrest at G₀-G₁ phase.     

Cyclin D- and E-Dependent Kinases and the p57KIP2 Inhibitor: Cooperative Interactions In Vivo

This study examines in vivo the role and functional interrelationships of components regulating exit from the G₁ resting phase into the DNA synthetic (S) phase of the cell cycle. Our approach made use of several key experimental attributes of the developing mouse lens, namely its strong dependence on pRb in maintenance of the postmitotic state, the down-regulation of cyclins D and E and up-regulation of the p57^KIP2 inhibitor in the postmitotic lens fiber cell compartment, and the ability to target transgene expression to this compartment. These attributes provide an ideal in vivo context in which to examine the consequences of forced cyclin expression and/or of loss of p57^KIP2 inhibitor function in a cellular compartment that permits an accurate quantitation of cellular proliferation and apoptosis rates in situ. Here, we demonstrate that, despite substantial overlap in cyclin transgene expression levels, D-type and E cyclins exhibited clear functional differences in promoting entry into S phase. In general, forced expression of the D-type cyclins was more efficient than cyclin E in driving lens fiber cells into S phase. In the case of cyclins D1 and D2, ectopic proliferation required their enhanced nuclear localization through CDK4 coexpression. High nuclear levels of cyclin E and CDK2, while not sufficient to promote efficient exit from G₁, did act synergistically with ectopic cyclin D/CDK4. The functional differences between D-type and E cyclins was most evident in the p57^KIP2-deficient lens wherein cyclin D overexpression induced a rate of proliferation equivalent to that of the pRb null lens, while overexpression of cyclin E did not increase the rate of proliferation over that induced by the loss of p57^KIP2 function. These in vivo analyses provide strong biological support for the prevailing view that the antecedent actions of cyclin D/CDK4 act cooperatively with cyclin E/CDK2 and antagonistically with p57^KIP2 to regulate the G₁/S transition in a cell type highly dependent upon pRb.     

Regulation of the cyclin D3 promoter by E2F1

We have previously demonstrated that ectopic expression of E2F1 is sufficient to drive quiescent cells into S phase and that E2F1 expression can contribute to oncogenic transformation. Key target genes in this process include master regulators of the cell cycle, such as cyclin E, which regulates G(1) progression, and cyclin A, which is required for the initiation of DNA synthesis. In the present work, we present novel evidence that a second G(1) cyclin, cyclin D3, is also potently activated by E2F1. First, an estrogen receptor-E2F1 fusion protein (ER-E2F1) potently activates the endogenous cyclin D3 mRNA upon treatment with 4-hydroxytamoxifen, which induces nuclear accumulation of the otherwise cytosolic fusion protein. Furthermore, trans-activation of cyclin D3 by ER-E2F1 occurs even in the presence of the protein synthesis inhibitor cycloheximide and thus appears direct. Second, all of the growth-stimulatory members of the E2F family (E2F1, -2, and -3A) potently activate a cyclin D3 promoter reporter, whereas growth-restraining members of the family (E2F4, -5, and -6) have little effect. Third, recombinant E2F1 binds with high affinity to the cyclin D3 promoter in vitro. Fourth, chromatin immunoprecipitation assays demonstrate that endogenous E2F1 is associated with the cyclin D3 promoter in vivo. Finally, mapping experiments localize the essential E2F regulatory element of the cyclin D3 promoter to a noncanonical E2F site in the promoter between nucleotides -143 and -135 relative to the initiating methionine codon. We conclude that in addition to cyclins E and A, E2F family members can also activate one member of the D-type cyclins, further contributing to the ability of the stimulatory E2F family members to drive cellular proliferation.     

Effects of a selectively bred novelty-seeking phenotype on the motivation to take cocaine in male and female rats

Background

Although colorectal cancer (CRC) is generally not considered to be a hormone-dependent malignancy, several sex-related differences in incidence, molecular characteristics and survival have been reported. Epidemiological studies have consistently shown that increased exposure to female sex hormones is associated with a lower risk of CRC in women, and cyclin D1, an important downstream effector in estrogen-mediated signaling, is commonly activated in CRC. In this study, we analyzed the prognostic significance of cyclin D1 expression in CRC, with particular reference to sex-related differences, in tumors from a large, prospective, population-based cohort.

Methods

Using tissue microarrays and immunohistochemistry, the fraction and intensity of cyclin D1 expression was evaluated in 527 incident CRC cases from the Malmö Diet and Cancer Study. The χ² and Spearman's rho (ρ) tests were used for comparison of cyclin D1 expression and relevant clinicopathological characteristics. Kaplan-Meier analysis and Cox proportional hazards modeling were used to assess the effect of cyclin D1 expression on cancer-specific survival (CSS) in univariate and multivariate analysis, adjusted for established prognostic factors.

Results

Cyclin D1 intensity was significantly lower in male compared with female CRC (P = 0.018). In the full cohort, cyclin D1 expression was associated with a significantly prolonged CSS (hazard ratio (HR) = 0.69; 95% CI 0.49 to 0.96, P = 0.026) but subgroup analysis according to gender revealed a strongly accentuated prognostic effect of cyclin D1 in male CRC (HR = 0.48; 95% CI 0.31 to 0.74, P < 0.001), which was in contrast to female CRC, where cyclin D1 was not prognostic (HR = 1.05; 95% CI 0.62 to 1.78, P = 0.864) (P _interaction = 0.024). The prognostic value of cyclin D1 was not retained in multivariate analysis, either in the full cohort or in male CRC.

Conclusions

Cyclin D1 expression is strongly associated with prolonged survival in male CRC. These findings not only support an important role for cyclin D1 in colorectal carcinogenesis, but also add further weight to the accumulating evidence that CRC is indeed a hormone-dependent malignancy, for which prognostic and treatment-predictive molecular biomarkers should be evaluated differently in women and men.     

Cyclin D1 Expression Mediated by Phosphatidylinositol 3-Kinase through mTOR-p70S6K-Independent Signaling in Growth Factor-Stimulated NIH 3T3 Fibroblasts

Phosphatidylinositol (PI) 3-kinase is required for G₁ to S phase cell cycle progression stimulated by a variety of growth factors and is implicated in the activation of several downstream effectors, including p70^S6K. However, the molecular mechanisms by which PI 3-kinase is engaged in activation of the cell cycle machinery are not well understood. Here we report that the expression of a dominant negative (DN) form of either the p110α catalytic or the p85 regulatory subunit of heterodimeric PI 3-kinase strongly inhibited epidermal growth factor (EGF)-induced upregulation of cyclin D1 protein in NIH 3T3(M17) fibroblasts. The PI 3-kinase inhibitors LY294002 and wortmannin completely abrogated increases in both mRNA and protein levels of cyclin D1 and phosphorylation of pRb, inducing G₁ arrest in EGF-stimulated cells. By contrast, rapamycin, which potently suppressed p70^S6K activity throughout the G₁ phase, had little inhibitory effect, if any, on either of these events. PI 3-kinase, but not rapamycin-sensitive pathways, was also indispensable for upregulation of cyclin D1 mRNA and protein by other mitogens in NIH 3T3 (M17) cells and in wild-type NIH 3T3 cells as well. We also found that an enforced expression of wild-type p110 was sufficient to induce cyclin D1 protein expression in growth factor-deprived NIH 3T3(M17) cells. The p110 induction of cyclin D1 in quiescent cells was strongly inhibited by coexpression of either of the PI 3-kinase DN forms, and by LY294002, but was independent of the Ras-MEK-ERK pathway. Unlike mitogen stimulation, the p110 induction of cyclin D1 was sensitive to rapamycin. These results indicate that the catalytic activity of PI 3-kinase is necessary, and could also be sufficient, for upregulation of cyclin D1, with mTOR signaling being differentially required depending upon cellular conditions.     

Oxidative stress-induced cyclin D1 depletion and its role in cell cycle processing

Background

Cyclin D1 is immediately down-regulated in response to reactive oxygen species (ROS) and implicated in the induction of cell cycle arrest in G2 phase by an unknown mechanism. Either treatment with a protease inhibitor alone or expression of protease-resistant cyclin D1 T286A resulted in only a partial relief from the ROS-induced cell cycle arrest, indicating the presence of an additional control mechanism.

Methods

Cells were exposed to hydrogen peroxide (H₂O₂), and analyzed to assess the changes in cyclin D1 level and its effects on cell cycle processing by kinase assay, de novo synthesis, gene silencing, and polysomal analysis, etc.

Results

Exposure of cells to excessive H₂O₂ induced ubiquitin-dependent proteasomal degradation of cyclin D1, which was subsequently followed by translational repression. This dual control mechanism was found to contribute to the induction of cell cycle arrest in G2 phase under oxidative stress. Silencing of an eIF2α kinase PERK significantly retarded cyclin D1 depletion, and contributed largely to rescuing cells from G2 arrest. Also the cyclin D1 level was found to be correlated with Chk1 activity.

Conlclusions

In addition to an immediate removal of the pre-existing cyclin D1 under oxidative stress, the following translational repression appear to be required for ensuring full depletion of cyclin D1 and cell cycle arrest. Oxidative stress-induced cyclin D1 depletion is linked to the regulation of G2/M transit via the Chk1–Cdc2 DNA damage checkpoint pathway.

General significance

The control of cyclin D1 is a gate keeping program to protect cells from severe oxidative damages.