Emerging evidence indicates that aldosterone and mineralocorticoid receptors (MRs) are associated with the pathogenesis of erectile dysfunction. However, the molecular mechanisms remain largely unknown. In this study, freshly isolated penile corpus cavernosum tissue from rats was treated with aldosterone, with or without MRs inhibitors. Nuclear factor (NF)-kappa B (NF-κB) activity was evaluated by real-time quantitative PCR, luciferase assay, and immunoblot. The results demonstrated that mRNA levels of the NF-κB target genes, including inhibitor of NF-κB alpha (IκB-α), NF-κB1, tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6), were higher after aldosterone treatment. Accordingly, phosphorylation of p65/RelA, IκB-α, and inhibitor of NF-κB kinase-β was markedly increased by aldosterone. Furthermore, knockdown of MRs prevented activation of the NF-κB canonical pathway by aldosterone. Consistent with this finding, ectopic overexpression of MRs enhanced the transcriptional activation of NF-κB by aldosterone. More importantly, the MRs antagonist, spironolactone blocked aldosterone-mediated activation of the canonical NF-κB pathway. In conclusion, aldosterone has an inflammatory effect in the corpus cavernosum penis, inducing NF-κB activation via an MRs-dependent pathway, which may be prevented by selective MRs antagonists. These data reveal the possible role of aldosterone in erectile dysfunction as well as its potential as a novel pharmacologic target for treatment.
Aldosterone/pharmacology* ; Animals ; Cytokines/biosynthesis* ; Gene Knockdown Techniques ; I-kappa B Kinase/antagonists & inhibitors* ; Interleukin-6/genetics* ; Male ; Mineralocorticoid Receptor Antagonists/pharmacology* ; NF-kappa B/genetics* ; Penis/metabolism* ; Protein Serine-Threonine Kinases/antagonists & inhibitors* ; RNA, Messenger/biosynthesis* ; Rats ; Rats, Inbred WKY ; Receptors, Mineralocorticoid/genetics* ; Signal Transduction/drug effects* ; Spironolactone/pharmacology* ; Transcriptional Activation ; Tumor Necrosis Factor-alpha/biosynthesis* ; NF-kappaB-Inducing Kinase

An F-box protein, beta-TrCP recognizes substrate proteins and destabilizes them through ubiquitin-dependent proteolysis. It regulates the stability of diverse proteins and functions as either a tumor suppressor or an oncogene. Although the regulation by beta-TrCP has been widely studied, the regulation of beta-TrCP itself is not well understood yet. In this study, we found that the level of beta-TrCP1 is downregulated by various protein kinase inhibitors in triple-negative breast cancer (TNBC) cells. A PI3K/mTOR inhibitor PI-103 reduced the level of beta-TrCP1 in a wide range of TNBC cells in a proteasome-dependent manner. Concomitantly, the levels of c-Myc and cyclin E were also downregulated by PI-103. PI-103 reduced the phosphorylation of beta-TrCP1 prior to its degradation. In addition, knockdown of beta-TrCP1 inhibited the proliferation of TNBC cells. We further identified that pharmacological inhibition of mTORC2 was sufficient to reduce the beta-TrCP1 and c-Myc levels. These results suggest that mTORC2 regulates the stability of beta-TrCP1 in TNBC cells and targeting beta-TrCP1 is a potential approach to treat human TNBC.
Cell Line, Tumor ; Cell Proliferation ; Cell Survival/drug effects ; Cyclin E/genetics/metabolism ; Dose-Response Relationship, Drug ; Female ; Furans/pharmacology ; Gene Knockdown Techniques ; Humans ; Models, Biological ; Multiprotein Complexes/antagonists & inhibitors ; Phosphatidylinositol 3-Kinases/*antagonists & inhibitors ; Phosphorylation/drug effects ; Protein Kinase Inhibitors/*pharmacology ; Proteolysis/drug effects ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; Pyridines/pharmacology ; Pyrimidines/pharmacology ; TOR Serine-Threonine Kinases/*antagonists & inhibitors ; Triple Negative Breast Neoplasms/genetics/*metabolism ; beta-Transducin Repeat-Containing Proteins/genetics/*metabolism

p70 ribosomal protein S6 kinase (p70S6K), an important member of AGC family, is a kind of multifunctional Ser/Thr kinases, which plays an important role in mTOR signaling cascade. The p70 ribosomal protein S6 kinase is closely associated with diverse cellular processes such as protein synthesis, mRNA processing, glucose homeostasis, cell growth and apoptosis. Recent studies have highlighted the important role of S6K in cancer, which arose interests of scientific researchers for the design and discovery of anti-cancer agents. Herein, the mechanisms of S6K and available inhibitors are reviewed.
Antineoplastic Agents ; Humans ; Protein Kinase Inhibitors ; chemistry ; Ribosomal Protein S6 Kinases, 70-kDa ; antagonists & inhibitors ; metabolism ; Signal Transduction ; TOR Serine-Threonine Kinases

We investigated how the dual inhibition of the molecular mechanism of the mammalian target of the rapamycin (mTOR) downstreams, P70S6 kinase (P70S6K) and eukaryotic initiation factor 4E (eIF4E), can lead to a suppression of the proliferation and progression of urothelial carcinoma (UC) in an orthotopic mouse non-muscle invasive bladder tumor (NMIBT) model. A KU-7-luc cell intravesically instilled orthotopic mouse NMIBC model was monitored using bioluminescence imaging (BLI) in vivo by interfering with different molecular components using rapamycin and siRNA technology. We then analyzed the effects on molecular activation status, cell growth, proliferation, and progression. A high concentration of rapamycin (10 microM) blocked both P70S6K and elF4E phosphorylation and inhibited cell proliferation in the KU-7-luc cells. It also reduced cell viability and proliferation more than the transfection of siRNA against p70S6K or elF4E. The groups with dual p70S6K and elF4E siRNA, and rapamycin reduced tumor volume and lamina propria invasion more than the groups with p70S6K or elF4E siRNA instillation, although all groups reduced photon density compared to the control. These findings suggest that both the mTOR pathway downstream of eIF4E and p70S6K can be successfully inhibited by high dose rapamycin only, and p70S6K and Elf4E dual inhibition is essential to control bladder tumor growth and progression.
Animals ; Cell Line ; Cell Proliferation/drug effects/genetics ; Cell Survival/drug effects ; Disease Progression ; Eukaryotic Initiation Factor-4E/*antagonists & inhibitors/genetics ; Female ; Mice ; Mice, Nude ; Mucous Membrane/pathology ; Phosphorylation/drug effects ; RNA Interference ; RNA, Small Interfering ; Ribosomal Protein S6 Kinases, 70-kDa/*antagonists & inhibitors/genetics ; Signal Transduction/drug effects ; Sirolimus/*pharmacology ; TOR Serine-Threonine Kinases/*antagonists & inhibitors/metabolism ; Urinary Bladder Neoplasms/genetics/*pathology ; Urothelium/pathology

We investigated how the dual inhibition of the molecular mechanism of the mammalian target of the rapamycin (mTOR) downstreams, P70S6 kinase (P70S6K) and eukaryotic initiation factor 4E (eIF4E), can lead to a suppression of the proliferation and progression of urothelial carcinoma (UC) in an orthotopic mouse non-muscle invasive bladder tumor (NMIBT) model. A KU-7-luc cell intravesically instilled orthotopic mouse NMIBC model was monitored using bioluminescence imaging (BLI) in vivo by interfering with different molecular components using rapamycin and siRNA technology. We then analyzed the effects on molecular activation status, cell growth, proliferation, and progression. A high concentration of rapamycin (10 microM) blocked both P70S6K and elF4E phosphorylation and inhibited cell proliferation in the KU-7-luc cells. It also reduced cell viability and proliferation more than the transfection of siRNA against p70S6K or elF4E. The groups with dual p70S6K and elF4E siRNA, and rapamycin reduced tumor volume and lamina propria invasion more than the groups with p70S6K or elF4E siRNA instillation, although all groups reduced photon density compared to the control. These findings suggest that both the mTOR pathway downstream of eIF4E and p70S6K can be successfully inhibited by high dose rapamycin only, and p70S6K and Elf4E dual inhibition is essential to control bladder tumor growth and progression.
Animals ; Cell Line ; Cell Proliferation/drug effects/genetics ; Cell Survival/drug effects ; Disease Progression ; Eukaryotic Initiation Factor-4E/*antagonists & inhibitors/genetics ; Female ; Mice ; Mice, Nude ; Mucous Membrane/pathology ; Phosphorylation/drug effects ; RNA Interference ; RNA, Small Interfering ; Ribosomal Protein S6 Kinases, 70-kDa/*antagonists & inhibitors/genetics ; Signal Transduction/drug effects ; Sirolimus/*pharmacology ; TOR Serine-Threonine Kinases/*antagonists & inhibitors/metabolism ; Urinary Bladder Neoplasms/genetics/*pathology ; Urothelium/pathology

OBJECTIVETo investigate the effects and mechanisms of cordycepin,an effective component of cordyceps militaris, on renal interstitial fibrosis (RIF) and its related eIF2α/TGF-β/Smad signaling pathway.

METHODFirstly, 15 C57BL/6 mice were randomly divided into 3 groups,the control group (Group A), the model group (Group B) and the cordycepin-treated group (Group C). After renal interstitial fibrotic model was successfully established by unilateral ureteral obstruction (UUO), the mice in Group C were intraperitoneally administrated with cordycepin(5 mg x kg(-1) d(-1)) and the ones in Group A and B were administrated with physiological saline for 5 days. At the end of the study, the obstructed kidneys were collected and detected for the pathological changes of RIF, and the mRNA expressions of collagen type I (Col I) and α-smooth muscle actin (α-SMA) in the kidney by Northern blot. Secondly, after renal tubular epithelial (NRK-52E) cells cultured in vitro were exposed to transforming growth factor (TGF) -β with or without cordycepin, the mRNA expressions of Col I and collagen type IV( Col IV) by Northern blot, and the protein expressions of eukaryotic initiation factor 2α (eIF2α), phosphorylated eIF2α ( p-eIF2α), Smad2/3 and phosphorylated Smad2/3 (p-Smad2/3) were tested by Western blot.

RESULTIn vivo, cordycepin alleviated RIF in model mice, including improving fibrotic pathological characteristics and mRNA expressions of Col I and α-SMA. In vitro, cordycepin induced the high expression of p-elF2α, and inhibited the expressions of p-Smad2/3, Col I and Col IV induced by TGF-β in NRK-52E cells.

CONCLUSIONCordycepin attenuates RIF in vivo and in vitro, probably by inducing the phosphorylation of eIF2α, suppressing the expression of p-Smad2/3, a key signaling molecule in TGF-β/Smad signaling pathway, and reducing the expressions of collagens and α-SMA in the kidney.

Actins ; analysis ; Animals ; Deoxyadenosines ; pharmacology ; Fibrosis ; Kidney ; drug effects ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Phosphorylation ; Protein-Serine-Threonine Kinases ; physiology ; Signal Transduction ; drug effects ; Smad Proteins ; physiology ; Transforming Growth Factor beta ; antagonists & inhibitors ; physiology

The expression of hypoxia-inducible factor (HIF) is influenced by reactive oxygen species (ROS). Effect of bilirubin on HIF-1 expression in proximal tubular cells was investigated under physiological oxygen concentration, which is relative hypoxic condition mimicking oxygen content in the medulla of renal tissue. The human kidney (HK2) cells were cultured in 5% oxygen with or without bilirubin. HIF-1alpha protein expression was increased by bilirubin treatment at 0.01-0.2 mg/dL concentration. The messenger RNA expression of HIF-1alpha was increased by 1.69+/-0.05 folds in the cells cultured with 0.1 mg/dL bilirubin, compared to the control cells. The inhibitors of PI3K/mTOR, PI3K/AKT, and ERK 1/2 pathways did not attenuate increased HIF-1alpha expression by bilirubin. HIF-1alpha expression decreased by 10 microM exogenous hydrogen peroxide (H2O2); scavenger of ROS with or without bilirubin in the HK2 cells increased HIF-1alpha concentration more than that in the cells without bilirubin. Exogenous H2O2 decreased the phosphorylation of P70S6 kinase, which was completely reversed by bilirubin treatment. Knockdown of NOX4 gene by small interfering RNA (siRNA) increased HIF-1alpha mRNA expression. In coonclusion, bilirubin enhances HIF-1alpha transcription as well as the up-regulation of HIF-1alpha protein translation through the attenuation of ROS and subunits of NADPH oxidase.
Bilirubin/*pharmacology ; Cell Line ; Epithelial Cells/cytology/metabolism ; Humans ; Hydrogen Peroxide/toxicity ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/*metabolism ; Kidney Tubules, Proximal/cytology ; Mitogen-Activated Protein Kinase 1/metabolism ; Mitogen-Activated Protein Kinase 3/metabolism ; NADPH Oxidase/antagonists & inhibitors/genetics/metabolism ; Oxygen/*pharmacology ; Phosphatidylinositol 3-Kinases/metabolism ; Phosphorylation/drug effects ; Proto-Oncogene Proteins c-akt/metabolism ; RNA Interference ; Ribosomal Protein S6 Kinases, 70-kDa/metabolism ; Signal Transduction/drug effects ; TOR Serine-Threonine Kinases/metabolism ; Transcriptional Activation/*drug effects ; Up-Regulation/drug effects

SARS coronavirus (SARS-CoV) develops an antagonistic mechanism by which to evade the antiviral activities of interferon (IFN). Previous studies suggested that SARS-CoV papain-like protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro efficiently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored PLpro domain (PLpro-TM) from SARS-CoV inhibits STING/TBK1/IKKε-mediated activation of type I IFNs and disrupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBK1. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKKε, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activation of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is disrupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3-TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which PLpro negatively regulates IRF3 activation by interaction with STING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.
Dimerization ; HEK293 Cells ; Humans ; I-kappa B Kinase ; metabolism ; Interferon Regulatory Factor-3 ; metabolism ; Interferon Type I ; antagonists & inhibitors ; metabolism ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Papain ; metabolism ; Peptide Hydrolases ; chemistry ; metabolism ; Phosphorylation ; Protein Binding ; Protein Structure, Tertiary ; Protein-Serine-Threonine Kinases ; metabolism ; SARS Virus ; enzymology ; Signal Transduction ; TNF Receptor-Associated Factor 3 ; metabolism ; Ubiquitination

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death in Korea. Curative treatment is only possible when the disease is diagnosed at the early stage. The prognosis of patients with HCC is even dismal in advanced stages. No systemic cytotoxic chemotherapy has proven to be beneficial in overall survival. Recently, the understanding of the molecular pathogenesis led to the development of new therapies. With the evidence of dysregulation of critical genes associated with cellular proliferation, growth factor signaling, cell cycling, apoptosis, and angiogenesis in HCC, a number of molecular target agents are under clinical trials. Sorafenib is the first systemic anticancer drug which has proven to gain survival benefit in the global as well as Asia-Pacific trials. However, the survival gain is still modest, and further efforts to improve outcomes in patients with HCC are necessary by developing novel drugs or combining other forms of therapies. This article will review signaling pathways in HCC and introduce molecular target agents under investigation currently.
Antineoplastic Agents/therapeutic use ; Carcinoma, Hepatocellular/*drug therapy/metabolism/pathology ; Humans ; Liver Neoplasms/*drug therapy/metabolism/pathology ; Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors/metabolism ; Molecular Targeted Therapy ; Niacinamide/analogs & derivatives/therapeutic use ; Phenylurea Compounds/therapeutic use ; Protein Kinase Inhibitors/therapeutic use ; Proto-Oncogene Proteins c-akt/antagonists & inhibitors/metabolism ; Receptor, IGF Type 1/antagonists & inhibitors/metabolism ; Signal Transduction ; TOR Serine-Threonine Kinases/antagonists & inhibitors/metabolism ; Wnt Proteins/antagonists & inhibitors/metabolism

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death in Korea. Curative treatment is only possible when the disease is diagnosed at the early stage. The prognosis of patients with HCC is even dismal in advanced stages. No systemic cytotoxic chemotherapy has proven to be beneficial in overall survival. Recently, the understanding of the molecular pathogenesis led to the development of new therapies. With the evidence of dysregulation of critical genes associated with cellular proliferation, growth factor signaling, cell cycling, apoptosis, and angiogenesis in HCC, a number of molecular target agents are under clinical trials. Sorafenib is the first systemic anticancer drug which has proven to gain survival benefit in the global as well as Asia-Pacific trials. However, the survival gain is still modest, and further efforts to improve outcomes in patients with HCC are necessary by developing novel drugs or combining other forms of therapies. This article will review signaling pathways in HCC and introduce molecular target agents under investigation currently.
Antineoplastic Agents/therapeutic use ; Carcinoma, Hepatocellular/*drug therapy/metabolism/pathology ; Humans ; Liver Neoplasms/*drug therapy/metabolism/pathology ; Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors/metabolism ; Molecular Targeted Therapy ; Niacinamide/analogs & derivatives/therapeutic use ; Phenylurea Compounds/therapeutic use ; Protein Kinase Inhibitors/therapeutic use ; Proto-Oncogene Proteins c-akt/antagonists & inhibitors/metabolism ; Receptor, IGF Type 1/antagonists & inhibitors/metabolism ; Signal Transduction ; TOR Serine-Threonine Kinases/antagonists & inhibitors/metabolism ; Wnt Proteins/antagonists & inhibitors/metabolism