Modulation Of Cell Cycle Regulators Research Articles

Circular RNAs in Cell Cycle Regulation of Cancers.

Cancer has been one of the most problematic health issues globally. Typically, all cancers share a common characteristic or cancer hallmark, such as sustaining cell proliferation, evading growth suppressors, and enabling replicative immortality. Indeed, cell cycle regulation in cancer is often found to be dysregulated, leading to an increase in aggressiveness. These dysregulations are partly due to the aberrant cellular signaling pathway. In recent years, circular RNAs (circRNAs) have been widely studied and classified as one of the regulators in various cancers. Numerous studies have reported that circRNAs antagonize or promote cancer progression through the modulation of cell cycle regulators or their associated signaling pathways, directly or indirectly. Mostly, circRNAs are known to act as microRNA (miRNA) sponges. However, they also hold additional mechanisms for regulating cellular activity, including protein binding, RNA-binding protein (RBP) recruitment, and protein translation. This review will discuss the current knowledge of how circRNAs regulate cell cycle-related proteins through the abovementioned mechanisms in different cancers.

In vitro and ex vivo anti-myeloma effects of nanocomposite As4S4/ZnS/Fe3O4

Nanoparticles in medicine can integrate actively targeted imaging agents and drug delivery vehicles, and combining multiple types of therapeutics in a single particle has numerous advantages, especially in multiple myeloma. MM is an incurable hematological disorder characterized by clonal proliferation of plasma cells in the bone marrow. In this study, we evaluated the anti-myeloma activity of 3 nanocomposites (3NPs): As4S4/ZnS/Fe3O4 (1:4:1), As4S4/ZnS/Fe3O4 with folic acid (FA), and As4S4/ZnS/Fe3O4 with FA and albumin with reduced survival MM cell lines and primary MM samples by each of 3NP. Cytotoxic effects of 3NPs were associated with caspase- and mitochondria-dependent apoptosis induction and reduced c-Myc expression. Modulation of cell cycle regulators, such as p-ATM/ATM and p-ATR/ATR, and increases in p-Chk2, cyclin B1, and histones were accompanied by G2/M arrest triggered by 3NPs. In addition, 3NPs activated several myeloma-related signaling, including JNK1/2/3, ERK1/2 and mTOR. To overcome BM microenvironment-mediated drug resistance, nanocomposites retained its anti-MM activity in the presence of stroma. 3NPs significantly decreased the stem cell-like side population in MM cells, even in the context of stroma. We observed strong synergistic effects of 3NPs combined with lenalidomide, pomalidomide, or melphalan, suggesting the potential of these combinations for future clinical studies.

TRPM7 Modulates Human Pancreatic Stellate Cell Activation.

Pancreatic diseases, such as pancreatitis or pancreatic ductal adenocarcinoma, are characterized by the presence of activated pancreatic stellate cells (PSCs). These cells represent key actors in the tumor stroma, as they actively participate in disease development and progression: reprograming these PSCs into a quiescent phenotype has even been proposed as a promising strategy for restoring the hallmarks of a healthy pancreas. Since TRPM7 channels have been shown to regulate hepatic stellate cells proliferation and survival, we aimed to study the role of these magnesium channels in PSC activation and proliferation. PS-1 cells (isolated from a healthy pancreas) were used as a model of healthy PSCs: quiescence or activation were induced using all-trans retinoic acid or conditioned media of pancreatic cancer cells, respectively. The role of TRPM7 was studied by RNA silencing or by pharmacological inhibition. TRPM7 expression was found to be correlated with the activation status of PS-1 cells. TRPM7 expression was able to regulate proliferation through modulation of cell cycle regulators and most importantly p53, via the PI3K/Akt pathway, in a magnesium-dependent manner. Finally, the analysis of TCGA database showed the overexpression of TRPM7 in cancer-associated fibroblasts. Taken together, we provide strong evidences that TRPM7 can be considered as a marker of activated PSCs.

Targeted Induction of Endogenous VDUP1 by Small Activating RNA Inhibits the Growth of Lung Cancer Cells.

Recent studies have reported that small double-strand RNAs (dsRNAs) can activate endogenous genes via an RNA-based promoter targeting mechanism termed RNA activation (RNAa). In the present study, we showed that dsVDUP1-834, a novel small activating RNA (saRNA) targeting promoter of vitamin D3 up-regulated protein 1 (VDUP1) gene, up-regulated expression of VDUP1 at both mRNA and protein levels in A549 lung cancer cells. We also demonstrated that dsVDUP1-834 inhibited cell proliferation in A549 lung cancer cells. Further studies showed that dsVDUP1-834 induced cell-cycle arrest by increasing p27 and p53 and decreasing cyclin A and cyclin B1. In addition, knockdown of VDUP1 abrogated dsVDUP1-834-induced up-regulation of VDUP1 gene expression and related effects. The activation of VDUP1 by dsVDUP1-834 was accompanied by an increase in dimethylation of histone 3 at lysine 4 (H3K4me2) and acetylation of histone 3 (H3ac) and a decrease in dimethylation of histone 3 at lysine 9 (H3K9me2) at the target site of VDUP1 promoter. Moreover, the enrichment of Ago2 was detected at the dsVDUP1-834 target site, and Ago2 knockdown significantly suppressed dsVDUP1-834-mediated inhibition of cell proliferation and modulation of cell-cycle regulators. Taken together, the results presented in this report demonstrate that dsVDUP1-834 induces VDUP1 gene expression by epigenetic changes, resulting in cell growth inhibition and cell-cycle arrest. Our results suggest that targeted induction of VDUP1 by dsVDUP1-834 might be a promising therapeutic strategy for the treatment of lung cancer.

Endocrine resistance in breast cancer: from molecular mechanisms to therapeutic strategies.

Estrogen receptor-positive (ER +) breast cancer accounts for approximately 75% of all breast cancers. Endocrine therapies, including selective ER modulators (SERMs), aromatase inhibitors (AIs), and selective ER down-regulators (SERDs) provide substantial clinical benefit by reducing the risk of disease recurrence and mortality. However, resistance to endocrine therapies represents a major challenge, limiting the success of ER + breast cancer treatment. Mechanisms of endocrine resistance involve alterations in ER signaling via modulation of ER (e.g., ER downregulation, ESR1 mutations or fusions); alterations in ER coactivators/corepressors, transcription factors (TFs), nuclear receptors and epigenetic modulators; regulation of signaling pathways; modulation of cell cycle regulators; stress signaling; and alterations in tumor microenvironment, nutrient stress, and metabolic regulation. Current therapeutic strategies to improve outcome of endocrine-resistant patients in clinics include inhibitors against mechanistic target of rapamycin (mTOR), cyclin-dependent kinase (CDK) 4/6, and the phosphoinositide 3-kinase (PI3K) subunit, p110α. Preclinical studies reveal novel therapeutic targets, some of which are currently tested in clinical trials as single agents or in combination with endocrine therapies, such as ER partial agonists, ER proteolysis targeting chimeras (PROTACs), next-generation SERDs, AKT inhibitors, epidermal growth factor receptor 1 and 2 (EGFR/HER2) dual inhibitors, HER2 targeting antibody-drug conjugates (ADCs) and histone deacetylase (HDAC) inhibitors. In this review, we summarize the established and emerging mechanisms of endocrine resistance, alterations during metastatic recurrence, and discuss the approved therapies and ongoing clinical trials testing the combination of novel targeted therapies with endocrine therapy in endocrine-resistant ER + breast cancer patients.

Anticancer activities of anthocyanin extract from genotyped Solanum tuberosum L. “Vitelotte”

The action of anthocyanins contained in the Vitelotte potato (Solanum tuberosum L.) in both breast and haematological cancers were investigated. The biomedical activities of anthocyanin extract derived from the Vitelotte cultivar were determined. Molecular genotyping was performed to properly identify this outstanding genotype in comparison to other potato varieties and to promote the utilization of this genetic resource by plant breeders. Furthermore, cellular and molecular characterization of the action of anthocyanin extract in cancer cells revealed that modulation of cell cycle regulators occurs upon treatment. As well as inducing apoptotic players such as TRAIL in cancer systems, anthocyanin extract inhibited Akt-mTOR signalling thereby inducing maturation of acute myeloid leukaemia cells. These results are of interest in view of the impact on food consumption and as functional food components on potential cancer treatment and prevention.

A stress-induced small RNA modulates alpha-rhizobial cell cycle progression.

Mechanisms adjusting replication initiation and cell cycle progression in response to environmental conditions are crucial for microbial survival. Functional characterization of the trans-encoded small non-coding RNA (trans-sRNA) EcpR1 in the plant-symbiotic alpha-proteobacterium Sinorhizobium meliloti revealed a role of this class of riboregulators in modulation of cell cycle regulation. EcpR1 is broadly conserved in at least five families of the Rhizobiales and is predicted to form a stable structure with two defined stem-loop domains. In S. meliloti, this trans-sRNA is encoded downstream of the divK-pleD operon. ecpR1 belongs to the stringent response regulon, and its expression was induced by various stress factors and in stationary phase. Induced EcpR1 overproduction led to cell elongation and increased DNA content, while deletion of ecpR1 resulted in reduced competitiveness. Computationally predicted EcpR1 targets were enriched with cell cycle-related mRNAs. Post-transcriptional repression of the cell cycle key regulatory genes gcrA and dnaA mediated by mRNA base-pairing with the strongly conserved loop 1 of EcpR1 was experimentally confirmed by two-plasmid differential gene expression assays and compensatory changes in sRNA and mRNA. Evidence is presented for EcpR1 promoting RNase E-dependent degradation of the dnaA mRNA. We propose that EcpR1 contributes to modulation of cell cycle regulation under detrimental conditions.

Plumbagin induces cell cycle arrest and autophagy and suppresses epithelial to mesenchymal transition involving PI3K/Akt/mTOR-mediated pathway in human pancreatic cancer cells.

Plumbagin (PLB), an active naphthoquinone compound, has shown potent anticancer effects in preclinical studies; however, the effect and underlying mechanism of PLB for the treatment of pancreatic cancer is unclear. This study aimed to examine the pancreatic cancer cell killing effect of PLB and investigate the underlying mechanism in human pancreatic cancer PANC-1 and BxPC-3 cells. The results showed that PLB exhibited potent inducing effects on cell cycle arrest in PANC-1 and BxPC-3 cells via the modulation of cell cycle regulators including CDK1/CDC2, cyclin B1, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53. PLB treatment concentration- and time-dependently increased the percentage of autophagic cells and significantly increased the expression level of phosphatase and tensin homolog, beclin 1, and the ratio of LC3-II over LC3-I in both PANC-1 and BxPC-3 cells. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B/mammalian target of rapamycin and p38 mitogen-activated protein kinase (p38 MAPK) pathways and activation of 5′-AMP-dependent kinase as indicated by their altered phosphorylation, contributing to the proautophagic activities of PLB in both cell lines. Furthermore, SB202190, a selective inhibitor of p38 MAPK, and wortmannin, a potent, irreversible, and selective PI3K inhibitor, remarkably enhanced PLB-induced autophagy in PANC-1 and BxPC-3 cells, indicating the roles of PI3K and p38 MAPK mediated signaling pathways in PLB-induced autophagic cell death in both cell lines. In addition, PLB significantly inhibited epithelial to mesenchymal transition phenotype in both cell lines with an increase in the expression level of E-cadherin and a decrease in N-cadherin. Moreover, PLB treatment significantly suppressed the expression of Sirt1 in both cell lines. These findings show that PLB promotes cell cycle arrest and autophagy but inhibits epithelial to mesenchymal transition phenotype in pancreatic cancer cells with the involvement of PI3K/protein kinase B/mammalian target of rapamycin and p38 MAPK mediated pathways.

Abstract 1884: Treatment with Hedgehog inhibitor PF-913 attenuates leukemia-initiation potential in acute myeloid leukemia cells

Abstract Aberrant activation of the Hedgehog (Hh) signaling pathway is involved in a variety of cancers, and required for maintenance of the leukemic stem cell (LSC) populations in several experimental systems. Cumulative evidence suggests that dormant self-renewing LSC contribute to acute myeloid leukemia (AML) propagation and relapse by evading conventional chemotherapies that target cycling cells. PF-04449913 (PF-913) is a novel oral small molecule inhibitor that selectively binds and targets Smoothened (SMO), a membrane protein regulating the Hh pathway. Treatment with PF-913 has shown promising results regarding safety, tolerability, and early signs of efficacy in patients with hematologic malignancies including AML. However, the detailed mechanisms and biomarkers remain to be elucidated in AML therapy. Using the co-culturing system with HS-5 stromal cells, the colony assay system, and the immunodeficient NOD/SCID/IL2rγnull (NOG) mouse model serially xenotransplanted with primary AML cells, we examined the effects of PF-913 on LSC population and AML propagation. In primary AML cells, the Hh signaling pathway was activated more in CD34-positive cells than CD34-negative cells. Ex vivo-treatment with PF-913 inhibited proliferation and induced minimal cell death in leukemia cell lines and primary AML cells. However, in vivo-treatment with PF-913 attenuated leukemia-initiation potential in AML cells through the serial transplantation system (0% human-CD45+ cells in bone marrow cells derived from the 2nd recipient mice), while limiting reduction of tumor burden in the primary leukemia system. Also in the colony-assay system using primary AML cells, treatment with PF-913 reduced serially colony formation. In leukemia cell lines and primary AML cells, treatment with PF-913 decreased the quiescent (Hoechst-33342low/Pyronin-Ylow) cell population as well as down-regulated mRNA encoding downstream effector GLIs and GLI-targeting molecules in the canonical Hh pathway. In the in vivo-NOG mouse system, comprehensive Gene Set Enrichment Analysis (GSEA) revealed that PF-913 treatment modulated cell cycle regulation and self-renewal signaling in primary AML cells. Moreover, combined treatment with PF-913 abrogated resistance to Ara-C in AML cell lines co-cultured with HS-5 stromal cells and sensitized AML cells to Ara-C in the cutaneous tumor system. Our findings imply that selective Hh inhibitor, PF-913 treatment can attenuate the leukemia-initiation potential in AML cells by modulation of cell cycle regulation and self-renewal signaling, and can also improve AML therapy through sensitizing dormant LSC to chemotherapy and overcoming the resistance in the bone marrow microenvironment. Citation Format: Yosuke Minami, Nobuaki Fukushima, Anil Sadarangani, Hironobu Minami, Catriona Jamieson, Tomoki Naoe. Treatment with Hedgehog inhibitor PF-913 attenuates leukemia-initiation potential in acute myeloid leukemia cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1884. doi:10.1158/1538-7445.AM2014-1884

Expression of neddylation-related proteins in melanoma cell lines and the effect of neddylation on melanoma proliferation.

Neddylation promotes the process of ubiquitination, which plays a critical role in the degradation of numerous proteins, including cell cycle and apoptosis regulators. In our previous study, an increase in neddylation was identified in melanoma cell lines. In the present study, the upregulation of neddylation was detected in melanoma tissues which confirmed the results of our previous study on melanoma cell lines. To explore the mechanism by which the process of neddylation was increased, the enzymes that regulate the process were investigated. These neddylation-related regulatory enzymes are potential targets for melanoma therapy. Downregulation of UBA3, a subunit of the E1 enzyme, by RNA interference caused cell cycle arrest at G0/G1 in the M14 cell line. In addition, cyclin D expression declined, whereas p27, p21 and bax expression increased. These findings suggest that interfering with the neddylation pathway may decrease the proliferation of melanoma through the modulation of cell cycle regulators and apoptosis promoters.

Treatment With Hedgehog Inhibitor, PF-04449913, Attenuates Leukemia-Initiation Potential In Acute Myeloid Leukemia Cells

BackgroundAberrant activation of the Hedgehog (Hh) signaling pathway is involved in a variety of cancers, and required for maintenance of the leukemic stem cell (LSC) populations in several experimental systems. Cumulative evidence suggests that dormant self-renewing LSC contribute to acute myeloid leukemia (AML) propagation and relapse by evading conventional chemotherapies that target cycling cells. PF-04449913 (PF) is a novel oral small molecule inhibitor that selectively binds and targets Smoothened (SMO), a membrane protein regulating the Hh pathway. Treatment with PF has shown promising Results regarding safety, tolerability, and early signs of efficacy in a phase 1 dose-escalation study of hematologic malignancies including AML (Jamieson C, et al. ASH, 2011). On the basis of these encouraging Results, phase 1b and phase 2 studies of PF in combination with chemotherapies have been planned in patients with AML. However, the detailed mechanisms and biomarkers remain to be elucidated in AML therapy with Hh pathway inhibitors. Research Aims and MethodsWe used AML cell lines and patient-derived primary AML cells in order to evaluate the efficacy and elucidate detailed mechanisms and biomarkers in the Hh antagonist, PF treatment. Using the co-culturing system with HS-5 stromal cells, the colony assay system, and the immunodeficient NOD/SCID/IL2rgnull (NOG) mouse model serially xenotransplanted with primary AML cells, we examined the effects of PF on LSC population and AML propagation. ResultsUsing FACS sorting and RQ-PCR assays of AML patient-derived primary cells, the Hh signaling pathway was activated more in CD34-positive cells than CD34-negative cells. Ex vivo-treatment with PF inhibited proliferation and induced minimal cell death in leukemia cell lines and primary AML cells. However, in vivo-treatment with PF attenuated leukemia-initiation potential in AML cells through the serial transplantation system, while limiting reduction of tumor burden in the primary leukemia system. Also in the colony-assay system using primary AML cells, treatment with PF reduced serially colony formation. In MOLM-14 cells, treatment with PF down-regulated mRNA encoding downstream effector GLIs and GLI-targeting molecules in the canonical Hh pathway using RQ-PCR assays, and decreased nuclear expression of GLI-2 using immunofluorescence assays. In addition, treatment with PF remarkably decreased the quiescent (Hoechst-33342low/Pyronin-Ylow) cell population and increased cycling cell population. In the in vivo-NOG mouse system, comprehensive Gene Set Enrichment Analysis (GSEA) revealed that PF treatment modulated cell cycle regulation and self-renewal signaling in primary AML cells. Moreover, combined treatment with PF abrogated resistance to Ara-C in AML cell lines co-cultured with HS-5 stromal cells and sensitized primary AML cells to Ara-C in the colony-assay system. We are also investigating toxicity for normal cord blood cells with PF treatment. ConclusionsOur findings imply that selective Hh inhibitor, PF treatment can attenuate the leukemia-initiation potential in AML cells by modulation of cell cycle regulation and self-renewal signaling, and can also improve AML therapy through sensitizing dormant LSC to chemotherapy and overcoming the resistance in the bone marrow microenvironment. Disclosures:Kiyoi:Kyowa Hakko Kirin Co. Ltd.: Research Funding; Novartis Pharma: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myers Squibb: Research Funding.

The HDAC interaction network

The HDAC interaction network

Abstract 18304: Identification of MicroRNAs Dysregulated in a Porcine Model of Coronary In-stent Restenosis

Introduction Vascular injury following stent delivery results in proliferation and migration of smooth muscle cells (SMC) leading to in-stent restenosis. MicroRNAs (miRs) are short, non-coding ribonucleic acids that act to control gene expression at a posttranscriptional level. MiR-21 and -221 positively regulate SMC proliferation through modulation of cell-cycle regulators. MiR-143 and -145 are important in the maintenance of vascular SMC in the differentiated state. Hypothesis We hypothesised that expression patterns of these key regulatory miRs in the vasculature would alter in response to stent-induced injury. Methods Pigs received standardized bare-metal (cobalt alloy) or drug-eluting (zotarolimus) stents (n=4/group), over-expanded to induce vessel injury, and were euthanized at 0, 7 or 28 days (BMS) and 28 days (DES). Total RNA was extracted from stented tissue. MiR expression levels were determined by quantitative real-time polymerase chain reaction and normalized to miR-199a-3p, a suitable endogenous control with mean cycle threshold + SD 25.70 + 0.84 across all samples. All comparisons made against control vessels (unstented arteries). Results MiR-21 levels were upregulated at 7 and 28 days in both stent types. No differences were seen between BMS and DES at 28 days. MiR-221 levels were elevated at Day 7 in the BMS group, returning to baseline at 28 days. MiR-145 levels showed immediate downregulation (Day 0) and stayed significantly low in all groups. MiR-143 levels did not show this immediate change, but were otherwise similar to miR-145. Levels of miR-145 only were significantly lower in arteries receiving DES compared with BMS at 28 days (75% vs. 48% downregulation). Conclusions In conclusion, vascular miRs show altered expression following stent insertion. MiR-145 levels were lower in arteries with DES, in comparison to BMS, at 28 days. This provides potentially important information for future manipulation of miRs in the vessel wall.

Potent activity of the Hsp90 inhibitor ganetespib in prostate cancer cells irrespective of androgen receptor status or variant receptor expression

Androgen ablation therapy represents the first line of therapeutic intervention in men with advanced or recurrent prostate tumors. However, the incomplete efficacy and lack of durable response to this clinical strategy highlights an urgent need for alternative treatment options to improve patient outcomes. Targeting the molecular chaperone heat shock protein 90 (Hsp90) represents a potential avenue for therapeutic intervention as its inhibition results in the coordinate blockade of multiple oncogenic signaling pathways in cancer cells. Moreover, Hsp90 is essential for the stability and function of numerous client proteins, a number of which have been causally implicated in the pathogenesis of prostate cancer, including the androgen receptor (AR). Here, we examined the preclinical activity of ganetespib, a small molecule inhibitor of Hsp90, in a panel of prostate cancer cell lines. Ganetespib potently decreased viability in all lines, irrespective of their androgen sensitivity or receptor status, and more effectively than the ansamycin inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). Interestingly, while ganetespib exposure decreased AR expression and activation, the constitutively active V7 truncated isoform of the receptor was unaffected by Hsp90 inhibition. Mechanistically, ganetespib exerted concomitant effects on mitogenic and survival pathways, as well as direct modulation of cell cycle regulators, to induce growth arrest and apoptosis. Further, ganetespib displayed robust antitumor efficacy in both AR-negative and positive xenografts, including those derived from the 22Rv1 prostate cancer cell line that co-expresses full-length and variant receptors. Together these data suggest that further investigation of ganetespib as a new therapeutic treatment for prostate cancer patients is warranted.

Modulation of cell cycle regulators by HDACs

Histone deacetylases (HDACs) catalyze the deacetylation of lysine residues on histones and non-histone proteins. HDACs have been shown to control the functions of key cell cycle proteins. Consistent with this, the overexpression of HDACs has been observed in multiple cancers, resulting in deregulation of the cell cycle and uncontrolled proliferation. This review focuses on the impact that HDACs have on cell cycle control through the deacetylation of proteins.

Flufenoxuron, an acylurea insect growth regulator, alters development of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) by modulating levels of chitin, soluble protein content, and HSP70 and p34 cdc2 in the larval tissues

The effect of sublethal concentrations, 0.00141% (LC 20), 0.00251% (LC 30), and 0.0036% (LC 40) of a dispersible formulation of an acylurea insect growth regulator, flufenoxuron (Cascade) on certain biochemical parameters in the larvae of Tribolium castaneum was investigated. When neonates were fed on diet treated with sublethal concentrations for 24 h, it was observed that at all concentrations tested, there was a significant reduction in chitin content on the 15th day of development. Total soluble protein content at LC 20 and LC 30 decreased with increasing age of the larvae. At LC 20 and LC 40 concentrations there was a progressive increase in the protein: chitin ratio as a function of increase in age of the larvae. SDS–PAGE analysis of the larval tissue extracts indicated gross quantitative changes in some of the protein bands (MW 50–97 kDa). Western blot analysis revealed significant increase in the level of HSP70 in the extracts of larvae fed on LC 30 treated diet, on the 7th and 10th day of development in the decreasing order. Conversely, a significant decrease in the hyper-phosphorylated form of p34 cdc2 kinase due to flufenoxuron treatment indicating modulation of cell cycle regulation was observed. Thus, sublethal concentrations of flufenoxuron alter expression of developmentally regulated proteins, HSP70 and p34 cdc2 and chitin formation in a stage-specific manner thereby resulting developmental abnormalities in T. castaneum.

The reaction of flavanols with nitrous acid protects against N-nitrosamine formation and leads to the formation of nitroso derivatives which inhibit cancer cell growth

Studies have suggested that diets rich in polyphenols such as flavonoids may lead to a reduced risk of gastrointestinal cancers. We demonstrate the ability of monomeric and dimeric flavanols to scavenge reactive nitrogen species derived from nitrous acid. Both epicatechin and dimer B2 (epicatechin dimer) inhibited nitrous acid-induced formation of 3-nitrotyrosine and the formation of the carcinogenic N-nitrosamine, N-nitrosodimethylamine. The reaction of monomeric and dimeric epicatechin with nitrous acid led to the formation of mono- and di-nitroso flavanols, whereas the reaction with hesperetin resulted primarily in the formation of nitrated products. Although, epicatechin was transferred across the jejunum of the small intestine yielding metabolites, its nitroso form was not absorbed. Dimer B2 but not epicatechin monomer inhibited the proliferation of, and triggered apoptosis in, Caco-2 cells. The latter was accompanied by caspase-3 activation and reductions in Akt phosphorylation, suggesting activation of apoptosis via inhibition of prosurvival signaling. Furthermore, the dinitroso derivative of dimer B2, and to a lesser extent the dinitroso-epicatechin, also induced significant toxic effects in Caco-2 cells. The inhibitory effects on cellular proliferation were paralleled by early inhibition of ERK 1/2 phosphorylation and later reductions in cyclin D1 levels, indicating modulation of cell cycle regulation in Caco-2 cells. These effects highlight multiple routes in which dietary derived flavanols may exert beneficial effects in the gastrointestinal tract.

Relationship and prognostic significance of phospho-(serine 166)-murine double minute 2 and Akt activation in node-negative breast cancer with regard to p53 expression

The Akt signalling pathway plays a central role in tumourigenesis. Activation of Akt is related to a more aggressive phenotype in various human cancers, including breast cancer. Its activation contributes to cancer progression via pleiotropic effects, including suppression of apoptosis and modulation of cell cycle regulation. Murine double minute 2 (MDM2) is an oncoprotein that inhibits the function of p53 tumour suppressor protein. Cell culture studies show that Akt-related phosphorylation of MDM2 at serine 166 allows MDM2 to gain nuclear entry and fulfil its p53 regulating function. This study was designed to analyse the relationship of phospho-MDM2 (pMDM2) expression with Akt activation to determine a possible prognostic relevance of pMDM2 in node-negative breast cancer with respect to Akt activation and p53 status. pMDM2, phospho-Akt (pAkt) and p53 protein expression status were analysed immunohistochemically in 121 paraffin-embedded breast cancer cases. Expression of pMDM2 correlated with Akt activation (P<0.001). Univariate analysis identified pMDM2 as a prognostic factor (P=0.0458) in node-negative breast cancers. The unfavourable prognostic significance was even more pronounced in tumours with a pMDM2(+)/pAkt(+) immunophenotype (P=0.0205). Stratification into a p53-negative subgroup further strengthened the adverse prognostic influence. These data confirm that MDM2 phosphorylation at serine 166 is mediated by Akt kinase. Besides the prognostic impact of pMDM2, our findings suggest that Akt-mediated modulation of the MDM2/p53 complex contributes to increased tumour aggressiveness especially in p53-negative breast cancers. However, due to the relatively small number of patients in this cohort, the results obtained need to be confirmed by larger cohorts.

Silibinin protects against photocarcinogenesis via modulation of cell cycle regulators, mitogen-activated protein kinases, and Akt signaling.

Here, we assessed the protective effect of silibinin on UVB-induced skin carcinogenesis in SKH-1 hairless mice. Topical application of silibinin before or immediately after UVB exposure or its dietary feeding resulted in a strong protection against photocarcinogenesis, in terms of tumor multiplicity (60-66%; P < 0.001), tumor volume per mouse (93-97%; P < 0.001) and tumor volume per tumor (80-91%; P < 0.001). Silibinin also moderately inhibited tumor incidence (5-15%; P < 0.01) and delayed tumor latency period (up to 4 weeks; P < 0.01-0.001). To investigate in vivo molecular mechanisms of silibinin efficacy, tumors and uninvolved skin from tumor-bearing mice were examined immunohistochemically for proliferation, p53, apoptosis, and activated caspase-3. Silibinin treatment showed a strong decrease (P < 0.001) in proliferating cell nuclear antigen-positive cells and an increase in p53-positive (P < 0.005-0.001), terminal deoxynucleotidyltransferase-mediated nick end labeling-positive (P < 0.005-0.001), and cleaved caspase-3-positive cells (P < 0.001). Western blot analysis of normal skin and tumor lysates showed that silibinin decreases the levels of cyclin-dependent kinase 2 and cyclin-dependent kinase 4 and associated cyclins A, E, and D1, together with an up-regulation of Cip1/p21, Kip1/p27, and p53. Silibinin also showed a strong phosphorylation of extracellular signal-regulated protein kinase 1/2, stress-activated protein kinase/c-JUN NH2-terminal kinase 1/2, and p38 mitogen-activated protein kinases but inhibited Akt phosphorylation and decreased survivin levels with an increase in cleaved caspase-3. Together, these results show a strong preventive efficacy of silibinin against photocarcinogenesis, which involves the inhibition of DNA synthesis, cell proliferation, and cell cycle progression and an induction of apoptosis. Furthermore, these results also identify in vivo molecular mechanisms of silibinin efficacy against photocarcinogenesis.

Dysregulation of the TGF-beta postreceptor signaling pathway in cell lines derived from primary or metastatic ovarian cancer.

Transforming growth factor-beta (TGF-beta) may cause cell cycle arrest, terminal differentiation, or apoptosis in most normal epithelial cells, whereas most malignant cell lines are resistant to TGF-beta. Mechanisms of resistance to TGF-beta caused by modulation of cell cycle regulators and/or inactivation of components of the TGF-beta signaling transduction pathway such as C-myc and Smad4 are not well understood. To investigate the potential association between loss of sensitivity to TGF-beta and expression status of transforming growth factor receptor II (TbetaR II) , Smad4, CDC25A and C-myc in 14 cell lines derived from ovarian cancer, the expression levels of these genes were detected by semi-quantitative RT-PCR. Normal ovarian surface tissues were used as controls. The expression of TbetaR II was detectable in all of 14 cell lines. The expression of Smad4 was decreased in 10 cell lines and 9 cell lines overexpressed CDC25A, as compared to normal controls. CDC25A gene was overexpressed with 88% (8/9) in tumorigenic cell lines as determined by xenografts in nude mice, and only in 20% (1/5) of non-tumorigenic cell lines (P<0.05). C-myc was not overexpressed in any of these cell lines. The loss of sensitivity to TGF-beta of cell lines derived from ovarian cancers may be related to a decreased expression of Smad4, which mediates TGF-beta induced growth inhibition, and/or an overexpression of CDC25A. This overexpression of CDC25A correlates with increased tumorigenicity of ovarian cancer cell lines. The loss of sensitivity to TGF-beta is not associated with a lack of TbetaR II.