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PubMed2003

Direct kinase-to-kinase signaling mediated by the FHA phosphoprotein recognition domain of the Dun1 DNA damage checkpoint kinase.

The serine-threonine kinase Dun1 contains a forkhead-associated (FHA) domain and functions in the DNA damage checkpoint pathway of Saccharomyces cerevisiae. It belongs to the Chk2 family of checkpoint kinases, which includes S. cerevisiae Rad53 and Mek1, Schizosaccharomyces pombe Cds1, and human Chk2. Dun1 is required for DNA damage-induced transcription of certain target genes, transient G(2)/M arrest after DNA damage, and DNA damage-induced phosphorylation of the DNA repair protein Rad55. Here we report that the FHA phosphoprotein recognition domain of Dun1 is required for direct phosphorylation of Dun1 by Rad53 kinase in vitro and in vivo. trans phosphorylation by Rad53 does not require the Dun1 kinase activity and is likely to involve only a transient interaction between the two kinases. The checkpoint functions of Dun1 kinase in DNA damage-induced transcription, G(2)/M cell cycle arrest, and Rad55 phosphorylation are severely compromised in an FHA domain mutant of Dun1. As a consequence, the Dun1 FHA domain mutant displays enhanced sensitivity to genotoxic stress induced by UV, methyl methanesulfonate, and the replication inhibitor hydroxyurea. We show that the Dun1 FHA domain is critical for direct kinase-to-kinase signaling from Rad53 to Dun1 in the DNA damage checkpoint pathway.

Cell Cycle↗

PubMed2004

Xenopus Cds1 is regulated by DNA-dependent protein kinase and ATR during the cell cycle checkpoint response to double-stranded DNA ends.

The checkpoint kinase Cds1 (Chk2) plays a key role in cell cycle checkpoint responses with functions in cell cycle arrest, DNA repair, and induction of apoptosis. Proper regulation of Cds1 is essential for appropriate cellular responses to checkpoint-inducing insults. While the kinase ATM has been shown to be important in the regulation of human Cds1 (hCds1), here we report that the kinases ATR and DNA-dependent protein kinase (DNA-PK) play more significant roles in the regulation of Xenopus Cds1 (XCds1). Under normal cell cycle conditions, nonactivated XCds1 constitutively associates with a Xenopus ATR complex. The association of XCds1 with this complex does not require a functional forkhead activation domain but does require a putative SH3 binding region that is found in XCds1. In response to double-stranded DNA ends, the amino terminus of XCds1 is rapidly phosphorylated in a sequential pattern. First DNA-PK phosphorylates serine 39, a site not previously recognized as important in Cds1 regulation. Xenopus ATM, ATR, and/or DNA-PK then phosphorylate three consensus serine/glutamine sites. Together, these phosphorylations have the dual function of inducing dissociation from the ATR complex and independently promoting the full activation of XCds1. Thus, the checkpoint-mediated activation of XCds1 requires phosphorylation by multiple phosphoinositide 3-kinase-related kinases, protein-protein dissociation, and autophosphorylation.

Amino Acid Sequence↗

PubMed2005

Mutations of the Yku80 C terminus and Xrs2 FHA domain specifically block yeast nonhomologous end joining.

The nonhomologous end-joining (NHEJ) pathway of DNA double-strand break repair requires three protein complexes in Saccharomyces cerevisiae: MRX (Mre11-Rad50-Xrs2), Ku (Ku70-Ku80), and DNA ligase IV (Dnl4-Lif1-Nej1). Much is known about the interactions that mediate the formation of each complex, but little is known about how they act together during repair. A comprehensive yeast two-hybrid screen of the NHEJ factors of S. cerevisiae revealed all known interactions within the MRX, Ku, and DNA ligase IV complexes, as well as three additional, weaker interactions between Yku80-Dnl4, Xrs2-Lif1, and Mre11-Yku80. Individual and combined deletions of the Yku80 C terminus and the Xrs2 forkhead-associated (FHA) domain were designed based on the latter two-hybrid results. These deletions synergistically blocked NHEJ but not the telomere and recombination functions of Ku and MRX, confirming that these protein regions are functionally important specifically for NHEJ. Further mutational analysis of Yku80 identified a putative C-terminal amphipathic alpha-helix that is both required for its NHEJ function and strikingly similar to a DNA-dependent protein kinase interaction motif in human Ku80. These results identify a novel role in yeast NHEJ for the poorly characterized Ku80 C-terminal and Xrs2 FHA domains, and they suggest that redundant binding of DNA ligase IV facilitates completion of this DNA repair event.

Antigens, Nuclear↗

PubMed1996

Mechanisms of gene expression and cell fate determination in the developing pulmonary epithelium.

The pulmonary epithelium is a derivative of the foregut endoderm. Proliferation and differentiation of the primitive pulmonary epithelium result in an array of epithelial cell phenotypes that determine lung function and the response of the lung to injury, infection, or neoplastic transformation. The establishment of a cell phenotype requires the presence of transcription factors that activate or repress expression of specific genes. Members of the forkhead family of transcription factors, in particular HNF-3 alpha, HNF-3 beta, HFH-4; the homeodomain protein TTF-1; and N-myc, are all expressed in the developing pulmonary epithelium and may play important regulatory roles during development. Two genes specific to the pulmonary epithelium, the surfactant protein A and Clara cell secretory protein genes, serve as useful paradigms for understanding the mechanisms regulating cell-specific gene expression in the pulmonary epithelium.

Amino Acid Sequence↗

PubMed2005

Alterations in growth and apoptosis of insulin receptor substrate-1-deficient beta-cells.

Insulin and IGF-I activate antiapoptotic pathways via insulin receptor substrate (IRS) proteins in most mammalian cells, including beta-cells. IRS-1 knockout (IRS-1KO) mice show growth retardation, hyperinsulinemia, and hyperplastic but dysfunctional islets without developing overt diabetes, whereas IRS-2KOs develop insulin resistance and islet hypoplasia leading to diabetes. Because both models display insulin resistance, it is difficult to differentiate islet response to insulin resistance from islet defects due to loss of proteins in the islets themselves. We used a transplantation approach, as a means of separating host insulin resistance from islet function, to examine alterations in proteins in insulin/IGF-I signaling pathways that may contribute to beta-cell proliferation and/or apoptosis in IRS-1KO islets. Islets isolated from wild-type (WT) or IRS-1KO mice were transplanted into WT or insulin-resistant IRS-1KO males under the kidney capsule. The beta-cell mitotic rate in transplanted islets in IRS-1KO recipients was increased 1.5-fold compared with WT recipients and was similar to that in endogenous pancreases of IRS-1KOs, whereas beta-cell apoptosis was reduced by approximately 80% in IRS-1KO grafts in IRS-1KO recipients compared with WT recipients. Immunohistochemistry showed a substantial increase in IRS-2 expression in IRS-1KO islets transplanted into IRS-1KO mice as well as in endogenous islets from IRS-1KOs. Furthermore, enhanced cytosolic forkhead transcription factor (FoxO1) staining in IRS-1KO grafts suggests intact Akt/PKB activity. Together, these data indicate that, even in the absence of insulin resistance, beta-cells deficient in IRS-1 exhibit a compensatory increase in IRS-2, which is associated with islet growth and is characterized by both proliferative and antiapoptotic effects that likely occur via an insulin/IGF-I/IRS-2 pathway.

Adaptation, Physiological↗

PubMed2006

The mRNA expression profile of metabolic genes relative to MHC isoform pattern in human skeletal muscles.

The metabolic profile of rodent muscle is generally reflected in the myosin heavy chain (MHC) fiber-type composition. The present study was conducted to test the hypothesis that metabolic gene expression is not tightly coupled with MHC fiber-type composition for all genes in human skeletal muscle. Triceps brachii, vastus lateralis quadriceps, and soleus muscle biopsies were obtained from normally physically active, healthy, young male volunteers, because these muscles are characterized by different fiber-type compositions. As expected, citrate synthase and 3-hydroxyacyl dehydrogenase activity was more than twofold higher in soleus and vastus than in triceps. Contrary, phosphofructokinase and total lactate dehydrogenase (LDH) activity was approximately three- and twofold higher in triceps than in both soleus and vastus. Expression of metabolic genes was assessed by determining the mRNA content of a broad range of metabolic genes. The triceps muscle had two- to fivefold higher MHC IIa, phosphofructokinase, and LDH A mRNA content and two- to fourfold lower MHC I, lipoprotein lipase, CD36, hormone-sensitive lipase, and LDH B and hexokinase II mRNA than vastus lateralis or soleus. Interestingly, such mRNA differences were not evident for any of the genes encoding mitochondrial oxidative proteins, 3-hydroxyacyl dehydrogenase, carnitine palmitoyl transferase I, citrate synthase, alpha-ketogluterate dehydrogenase, and cytochrome c, nor for the transcriptional regulators peroxisome proliferator activator receptor gamma coactivator-1alpha, forkhead box O1, or peroxisome proliferator activator receptor-alpha. Thus the mRNA expression of genes encoding mitochondrial proteins and transcriptional regulators does not seem to be fiber type specific as the genes encoding glycolytic and lipid metabolism genes, which suggests that basal mRNA regulation of genes encoding mitochondrial proteins does not match the wide differences in mitochondrial content of these muscles.

Adult↗

PubMed2004

Pyruvate dehydrogenase activation and kinase expression in human skeletal muscle during fasting.

Fasting forces adaptive changes in whole body and skeletal muscle metabolism that increase fat oxidation and decrease the oxidation of carbohydrate. We tested the hypothesis that 40 h of fasting would decrease pyruvate dehydrogenase (PDH) activity and increase PDH kinase (PDK) isoform mRNA expression in human skeletal muscle. The putative transcriptional activators of PDK isozymes, peroxisome proliferator-activated receptor-alpha (PPAR-alpha) protein, and forkhead homolog in rhabdomyosarcoma (FKHR) mRNA were also measured. Eleven healthy adults fasted after a standard meal (25% fat, 60% carbohydrate, 15% protein) with blood and skeletal muscle samples taken at 3, 15, and 40 h postprandial. Fasting increased plasma free fatty acid, glycerol, and beta-hydroxybutyrate concentrations and decreased glucose and insulin concentrations. PDH activity decreased from 0.88 +/- 0.11 mmol acetyl-CoA. min(-1). kg wet muscle wt(-1) at 3 h to 0.62 +/- 0.10 (P = not significant) and 0.39 +/- 0.06 (P < 0.05) mmol. min(-1). kg wet mass(-1) after 15 and 40 h of fasting. Although all four PDK isoforms were expressed in human skeletal muscle, PDK-2 and -4 mRNA were the most abundant. PDK-1 and -3 mRNA abundance was approximately 1 and 15% of the PDK-2 and -4 levels, respectively. The 40-h fast had no effect on PDK-1, -2, and -3 mRNA expression. PDK-4 mRNA was significantly increased approximately 3-fold after 15 h and approximately 14-fold after 40 h of fasting. Skeletal muscle PPAR-alpha protein and FKHR mRNA abundance were unaffected by the fast. The results suggest that decreased PDH activation after 40 h of fasting may have been a function of the large increase in PDK-4 mRNA expression and possible subsequent increase in PDK protein and activity. The changes in PDK-4 expression and PDH activity did not coincide with increases in the transcriptional activators PPAR-alpha and FKHR.

Adult↗

PubMed2003

Alterations of gene expression in failing myocardium following left ventricular assist device support.

Chronic unloading of the failing heart with a left ventricular assist device (LVAD) can decrease cardiac mass and myocyte size and has the potential to improve contractile function. To study the effect of chronic ventricular unloading on myocardial gene expression, a microarray (U133A, Affymetrix) profiling gene expression was compared before and after LVAD support in seven patients with idiopathic dilated cardiomyopathy and end-stage heart failure. On average, 1,374 +/- 155 genes were reported as "increased" and 1,629 +/- 45 as "decreased" after LVAD support. A total of 130 gene transcripts achieved the strict criteria for upregulation and 49 gene transcripts for downregulation after LVAD support. Upregulated genes included a large proportion of transcription factors, genes related to cell growth/apoptosis/DNA repair, cell structure proteins, metabolism, and cell signaling/communication. LVAD support resulted in downregulation of genes for a group of cytokines. To validate the array data, 10 altered genes were confirmed by real-time RT-PCR. Further study showed that the phosphoinositide-3-kinase-forkhead protein pathway and proteins related to nitric oxide synthesis, including eNOS and dimethylarginine dimethylaminohydrolase isoform 1 (DDAH1, an enzyme regulating endogenous nitric oxide synthase activity), were significantly increased during the cardiac remodeling process. Increased eNOS and DDAH1 expression after LVAD support may contribute to improved endothelial function of the failing hearts.

Adult↗

PubMed2006

(Patho)physiological significance of the serum- and glucocorticoid-inducible kinase isoforms.

The serum- and glucocorticoid-inducible kinase-1 (SGK1) is ubiquitously expressed and under genomic control by cell stress (including cell shrinkage) and hormones (including gluco- and mineralocorticoids). Similar to its isoforms SGK2 and SGK3, SGK1 is activated by insulin and growth factors via phosphatidylinositol 3-kinase and the 3-phosphoinositide-dependent kinase PDK1. SGKs activate ion channels (e.g., ENaC, TRPV5, ROMK, Kv1.3, KCNE1/KCNQ1, GluR1, GluR6), carriers (e.g., NHE3, GLUT1, SGLT1, EAAT1-5), and the Na+-K+-ATPase. They regulate the activity of enzymes (e.g., glycogen synthase kinase-3, ubiquitin ligase Nedd4-2, phosphomannose mutase-2) and transcription factors (e.g., forkhead transcription factor FKHRL1, beta-catenin, nuclear factor kappaB). SGKs participate in the regulation of transport, hormone release, neuroexcitability, cell proliferation, and apoptosis. SGK1 contributes to Na+ retention and K+ elimination of the kidney, mineralocorticoid stimulation of salt appetite, glucocorticoid stimulation of intestinal Na+/H+ exchanger and nutrient transport, insulin-dependent salt sensitivity of blood pressure and salt sensitivity of peripheral glucose uptake, memory consolidation, and cardiac repolarization. A common ( approximately 5% prevalence) SGK1 gene variant is associated with increased blood pressure and body weight. SGK1 may thus contribute to metabolic syndrome. SGK1 may further participate in tumor growth, neurodegeneration, fibrosing disease, and the sequelae of ischemia. SGK3 is required for adequate hair growth and maintenance of intestinal nutrient transport and influences locomotive behavior. In conclusion, the SGKs cover a wide variety of physiological functions and may play an active role in a multitude of pathophysiological conditions. There is little doubt that further targets will be identified that are modulated by the SGK isoforms and that further SGK-dependent in vivo physiological functions and pathophysiological conditions will be defined.

Animals↗

PubMed2004

Autoantibodies to Annexin XI-A and Other Autoantigens in the Diagnosis of Breast Cancer.

We report on the identification of autoantigens commonly recognized by sera from patients with breast cancer. We selected ten sera from patients with invasive ductal carcinoma (IDC) of the breast with high titer IgG autoantibodies for biopanning of a T7 phage breast cancer cDNA display library. A high throughput method involved the assembly of 938 T7 phages encoding potential breast cancer autoantigens. Microarrays of positive phages were probed with sera from 90 patients with breast cancer [15 patients with ductal carcinoma in situ (DCIS) and 75 patients with IDC of the breast], with 51 non-cancer control sera and with sera from 21 patients with systemic autoimmune diseases. A 12-phage breast cancer predictor group was constructed with phage inserts recognized by sera from patients with breast cancer and not by non-cancer or autoimmune control sera (P < 0.0001). Several autoantigens including annexin XI-A, the p80 subunit of the Ku antigen, ribosomal protein S6, and other unknown autoantigens could significantly discriminate between breast cancer and non-cancer control sera. Biopanning with three different sera led to the cloning of partial cDNA sequences identical to annexin XI-A. IgG autoantibodies reacting with the amino acid 41-74 sequence of annexin XI-A were found in 19% of all women with breast cancer but in 60% of sera from women with DCIS of the breast. In addition, partial sequences identical to annexin XI-A, nucleolar protein interacting with the forkhead-associated (FHA) domain of pKi-67, the KIAA1671 gene product, ribosomal protein S6, cyclin K, elongation factor-2, Grb2-associated protein 2, and other unknown proteins could distinguish DCIS from IDC of the breast and appear to be potential biomarkers for the diagnosis of breast cancer.

Annexins↗

PubMed2004

Inhibition of akt/protein kinase B signaling by naltrindole in small cell lung cancer cells.

The phosphatidylinositol 3-kinase-Akt/protein kinase B (PKB) survival signaling is very important for cancer cell survival and growth. Constitutively active phosphatidylinositol 3-kinase-Akt/PKB signaling in small cell lung cancer (SCLC) is a major factor for the survival of SCLC cells. Inhibitors of this signaling pathway would be potential antitumor agents, particularly for SCLC. Here we report that naltrindole, which has been used as a classic delta opioid antagonist, inhibited growth and induced apoptosis in the three characteristic SCLC cell lines, NCI-H69, NCI-H345, and NCI-H510. Naltrindole treatment reduced constitutive phosphorylation of Akt/PKB on serine 473 and threonine 308 in cells. We found that the levels of constitutive phosphorylation of Akt/PKB on serine 473 correlate with the sensitivity of the three cell lines to naltrindole treatment. Furthermore, naltrindole treatment not only reduced the phosphorylation of the Akt/PKB upstream kinase phosphoinositide-dependent kinase-1, but also its downstream effectors glycogen synthase kinase-3beta and the Forkhead transcription factors AFX and FKHR. DNA array analysis of 205 apoptosis-related genes indicated that some Akt/PKB-dependent genes were either up- or down-regulated by naltrindole. Flow cytometric and microscopic analyses clearly showed that naltrindole induced apoptosis in SCLC cells. RNA interference experiments confirmed that naltrindole-induced cell death was associated with the Akt/PKB survival pathway. Together, these results show that naltrindole is a new inhibitor of the Akt/PKB signaling pathway, suggesting that naltrindole could be a potential lead for the development of a new type of inhibitors that target the constitutively active Akt/PKB signaling-dependent SCLC cells.

Apoptosis↗

PubMed2004

Aberrant expression of the tyrosine kinase receptor EphA4 and the transcription factor twist in Sézary syndrome identified by gene expression analysis.

Sézary syndrome (Sz) is a malignancy of CD4+ memory skin-homing T cells and presents with erythroderma, lymphadenopathy, and peripheral blood involvement. To gain more insight into the molecular features of Sz, oligonucleotide array analysis was performed comparing gene expression patterns of CD4+ T cells from peripheral blood of patients with Sz with those of patients with erythroderma secondary to dermatitis and healthy controls. Using unsupervised hierarchical clustering gene, expression patterns of T cells from patients with Sz were classified separately from those of benign T cells. One hundred twenty-three genes were identified as significantly differentially expressed and had an average fold change exceeding 2. T cells from patients with Sz demonstrated decreased expression of the following hematopoietic malignancy-linked tumor suppressor genes: TGF-beta receptor II, Mxi1, Riz1, CREB-binding protein, BCL11a, STAT4, and Forkhead Box O1A. Moreover, the tyrosine kinase receptor EphA4 and the potentially oncogenic transcription factor Twist were highly and selectively expressed in T cells of patients with Sz. High expression of EphA4 and Twist was also observed in lesional skin biopsy specimens of a subset of patients with cutaneous T cell lymphomas related to Sz, whereas their expression was nearly undetectable in benign T cells or in skin lesions of patients with inflammatory dermatoses. Detection of EphA4 and Twist may be used in the molecular diagnosis of Sz and related cutaneous T-cell lymphomas. Furthermore, the membrane-bound EphA4 receptor may serve as a target for directed therapeutic intervention.

Female↗

PubMed2005

Gene expression profiles in pancreatic intraepithelial neoplasia reflect the effects of Hedgehog signaling on pancreatic ductal epithelial cells.

Invasive pancreatic cancer is thought to develop through a series of noninvasive duct lesions known as pancreatic intraepithelial neoplasia (PanIN). We used cDNA microarrays interrogating 15,000 transcripts to identify 49 genes that were differentially expressed in microdissected early PanIN lesions (PanIN-1B/2) compared with microdissected normal duct epithelium. In this analysis, a cluster of extrapancreatic foregut markers, including pepsinogen C, MUC6, KLF4, and TFF1, was found to be up-regulated in PanIN. Up-regulation of these genes was further validated using combinations of real-time reverse transcription-PCR, in situ hybridization, and immunohistochemistry in a total of 150 early PanIN lesions from 81 patients. Identification of these gastrointestinal transcripts in human PanIN prompted assessment of other foregut markers by both semiquantitative and real-time reverse transcription-PCR, revealing similar up-regulation of Sox-2, Gastrin, HoxA5, GATA4/5/6, Villin and Forkhead 6 (Foxl1). In contrast to frequent expression of multiple gastric epithelial markers, the intestinal markers intestinal fatty acid binding protein, CDX1 and CDX2 were rarely expressed either in PanIN lesions or in invasive pancreatic cancer. Hedgehog pathway activation induced by transfection of immortalized human pancreatic ductal epithelial cells with Gli1 resulted in up-regulation of the majority of foregut markers seen in early PanIN lesions. These data show frequent up-regulation of foregut markers in early PanIN lesions and suggest that PanIN development may involve Hedgehog-mediated conversion to a gastric epithelial differentiation program.

Biomarkers, Tumor↗

PubMed2005

Functional characterization of EBV-encoded nuclear antigen 1-specific CD4+ helper and regulatory T cells elicited by in vitro peptide stimulation.

CD4(+) helper and regulatory T (Treg) cells play important but opposing roles in regulating host immune responses against cancer and other diseases. However, very little is known about the antigen specificity of CD4(+) Treg cells. Here we describe the generation of a panel of EBV-encoded nuclear antigen 1 (EBNA1)-specific CD4(+) T-cell lines and clones that recognize naturally processed EBNA1-P(607-619) and -P(561-573) peptides in the context of HLA-DQ2 and HLA-DR11, -DR12, and -DR13 molecules, respectively. Phenotypic and functional analyses of these CD4(+) T cells revealed that they represent EBNA1-specific CD4(+) T helper as well as Treg cells. CD4(+) Treg cells do not secrete interleukin (IL)-10 and transforming growth factor beta cytokines but express CD25, the glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), and Forkhead Box P3 (Foxp3), and are capable of suppressing the proliferative responses of naive CD4(+) and CD8(+) T cells to stimulation with mitogenic anti-CD3 antibody. The suppressive activity of these CD4(+) Treg cells is mediated via cell-cell contact or in part by a cytokine-dependent manner. Importantly, these Treg cells suppress IL-2 secretion by CD4(+) effector T cells specific for either EBNA1 or a melanoma antigen, suggesting that these CD4(+) Treg cells induce immune suppression. These observations suggest that the success of peptide-based vaccines against EBV-associated cancer and other diseases may likely depend upon our ability to identify antigens/peptides that preferentially activate helper T cells and/or to design strategies to regulate the balance between CD4(+) helper and Treg cells.

Amino Acid Sequence↗

PubMed2006

The phosphoinositide 3-kinase/Akt pathway: a new target in human renal cell carcinoma therapy.

Metastatic renal cell carcinoma is resistant to current therapies. The phosphoinositide 3-kinase (PI3K)/Akt signaling cascade induces cell growth, cell transformation, and neovascularization. We evaluated whether targeting this pathway could be of therapeutic value against human renal cell carcinoma. The activation of the PI3K/Akt pathway and its role in renal cell carcinoma progression was evaluated in vitro in seven human cell lines by Western blot, cell counting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, terminal deoxyribonucleotide transferase-mediated nick-end labeling assays, and fluorescence-activated cell sorting analysis, using two PI3K inhibitors, LY294002 and wortmannin, as well as by transfection with various Akt constructs and through Akt knockdown by small interfering RNA (siRNA). In vivo nude mice bearing human renal cell carcinoma tumor xenografts were treated with LY294002 (75 mg/kg/wk, 4 weeks, i.p.). Tumor growth was measured and tumors were subjected to Western blot and immunohistochemical analysis. Akt was constitutively activated in all cell lines. Constitutive phosphorylation of glycogen synthase kinase-3 (GSK-3) was observed in all cell lines, whereas forkhead transcription factor and mammalian target of rapamycin, although expressed, were not constitutively phosphorylated. Exposure to LY294002 or wortmannin decreased Akt activation and GSK-3 phosphorylation and reduced cell growth by up to 70% through induction of cell apoptosis. These effects were confirmed by transfection experiments with Akt constructs or Akt siRNA. Importantly, LY294002 induced up to 50% tumor regression in mice through tumor cell apoptosis. Tumor neovascularization was significantly increased by LY294002 treatment. Blood chemistries showed no adverse effects of the treatment. Our results suggest an important role of PI3K/Akt inhibitors as a potentially useful treatment for patients with renal cell carcinoma.

Animals↗

PubMed2005

Loss of phosphatase and tensin homologue increases transforming growth factor beta-mediated invasion with enhanced SMAD3 transcriptional activity.

In normal epithelial tissues, the multifunctional cytokine transforming growth factor-beta (TGF-beta) acts as a tumor suppressor through growth inhibition and induction of differentiation whereas in advanced cancers, TGF-beta promotes tumor progression through induction of tumor invasion, neoangiogenesis, and immunosuppression. The molecular mechanisms through which TGF-beta shifts from a tumor suppressor to a tumor enhancer are poorly understood. We now show a role for the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in repressing the protumorigenic effects of TGF-beta. The TGF-beta effector SMAD3 inducibly interacts with PTEN on TGF-beta treatment under endogenous conditions. RNA interference (RNAi) suppression of PTEN expression enhances SMAD3 transcriptional activity and TGF-beta-mediated induction of SMAD3 target genes whereas reconstitution of PTEN in a null cancer cell line represses the expression of TGF-beta-regulated target genes. Targeting PTEN expression through RNAi in a PTEN wild-type cell line increases TGF-beta-mediated invasion but does not affect TGF-beta-mediated growth inhibition. Reconstitution of PTEN expression in a PTEN-null cell line blocks TGF-beta-induced invasion but does not modulate TGF-beta-mediated growth regulation. These effects are distinct from Akt and Forkhead family members that also interact with SMAD3 to regulate apoptosis or proliferation, respectively. Pharmacologic inhibitors targeting TGF-beta receptors and phosphatidylinositol 3-kinase signaling downstream from PTEN cooperate to block TGF-beta-mediated invasion. Thus, the loss of PTEN expression in human cancers may contribute to a role for TGF-beta as a tumor enhancer with specific effects on cellular motility and invasion.

Cell Movement↗

PubMed2005

Pentameric procyanidin from Theobroma cacao selectively inhibits growth of human breast cancer cells.

A naturally occurring, cocoa-derived pentameric procyanidin (pentamer) was previously shown to cause G0/G1 cell cycle arrest in human breast cancer cells by an unknown molecular mechanism. Here, we show that pentamer selectively inhibits the proliferation of human breast cancer cells (MDA MB-231, MDA MB-436, MDA MB-468, SKBR-3, and MCF-7) and benzo(a)pyrene-immortalized 184A1N4 and 184B5 cells. In contrast, normal human mammary epithelial cells in primary culture and spontaneously immortalized MCF-10A cells were significantly resistant. We evaluated whether this differential response to pentamer may involve depolarization of the mitochondrial membrane. Pentamer caused significant depolarization of mitochondrial membrane in MDA MB231 cells but not the more normal MCF-10A cells, whereas other normal and tumor cell lines tested gave variable results. Further investigations, using a proteomics approach with pentamer-treated MDA MB-231, revealed a specific dephosphorylation, without changes in protein expression, of several G1-modulatory proteins: Cdc2 (at Tyr15), forkhead transcription factor (at Ser256, the Akt phosphorylation site) and p53 (Ser392). Dephosphorylation of p53 (at Ser392) by pentamer was confirmed in MDA MB-468 cells. However, both expression and phosphorylation of retinoblastoma protein were decreased after pentamer treatment. Our results show that breast cancer cells are selectively susceptible to the cytotoxic effects of pentameric procyanidin, and suggest that inhibition of cellular proliferation by this compound is associated with the site-specific dephosphorylation or down-regulation of several cell cycle regulatory proteins.

Antioxidants↗

PubMed2006

Plumbagin induces G2-M arrest and autophagy by inhibiting the AKT/mammalian target of rapamycin pathway in breast cancer cells.

This study is the first to investigate the anticancer effect of plumbagin in human breast cancer cells. Plumbagin exhibited cell proliferation inhibition by inducing cells to undergo G2-M arrest and autophagic cell death. Blockade of the cell cycle was associated with increased p21/WAF1 expression and Chk2 activation, and reduced amounts of cyclin B1, cyclin A, Cdc2, and Cdc25C. Plumbagin also reduced Cdc2 function by increasing the association of p21/WAF1/Cdc2 complex and the levels of inactivated phospho-Cdc2 and phospho-Cdc25C by Chk2 activation. Plumbagin triggered autophagic cell death but not predominantly apoptosis. Pretreatment of cells with autophagy inhibitor bafilomycin suppressed plumbagin-mediated cell death. We also found that plumbagin inhibited survival signaling through the phosphatidylinositol 3-kinase/AKT signaling pathway by blocking the activation of AKT and downstream targets, including the mammalian target of rapamycin, forkhead transcription factors, and glycogen synthase kinase 3beta. Phosphorylation of both of mammalian target of rapamycin downstream targets, p70 ribosomal protein S6 kinase and 4E-BP1, was also diminished. Overexpression of AKT by AKT cDNA transfection decreased plumbagin-mediated autophagic cell death, whereas reduction of AKT expression by small interfering RNA potentiated the effect of plumbagin, supporting the inhibition of AKT being beneficial to autophagy. Furthermore, suppression of AKT by plumbagin enhanced the activation of Chk2, resulting in increased inactive phosphorylation of Cdc25C and Cdc2. Further investigation revealed that plumbagin inhibition of cell growth was also evident in a nude mouse model. Taken together, these results imply a critical role for AKT inhibition in plumbagin-induced G2-M arrest and autophagy of human breast cancer cells.

Animals↗