Check out our New Publishers' Select for Free Articles

Biochemical Pharmacology (v.85, #7)

Editorial Board (pp. iii).

BMP signaling in development and diseases: A pharmacological perspective by Amitabha Bandyopadhyay; Prem Swaroop Yadav; Paritosh Prashar (pp. 857-864).

Bone morphogenetic protein (BMP) signaling has been implicated in several processes during embryonic development and in adult tissue homeostasis. Maintenance of many organs such as skin, intestinal villi, bones and bone marrow requires continuous regeneration and subsequent differentiation of stem cells in order to maintain organ shape and size necessary for proper functioning. Although BMPs were initially identified as osteogenic factors present in demineralized bone capable of inducing ectopic bone formation, it is now evident that BMPs perform several other functions during embryonic development as well as during the adult life of an organism. Many disorders have been linked to either the BMPs or the molecules functioning downstream of BMP signaling pathway. This review summarizes the existing literature describing the role of BMP signaling during embryonic development and in adult tissue homeostasis to provide a perspective on pharmacological interventions of BMP signaling pathway to mitigate several disease conditions.

Keywords: BMP; Wnt; FGF; Cartilage; Bone

Toxico-pharmacological perspective of the Nrf2-Keap1 defense system against oxidative stress in kidney diseases by Hideyuki Saito (pp. 865-872).

The Nrf2-Keap1 defense pathway and oxidative stress-inducing factors in renal tubular cells in association with kidney diseases or injuries.Oxidative stress, including the generation of reactive oxygen species (ROS), appears to be responsible for the high incidence of cardiovascular events in patients with chronic kidney disease (CKD), and for the progression of CKD to end-stage renal disease. The processes for oxidative stress include increased generation and decreased elimination of ROS that could be caused by an impaired antioxidant defense system. Nuclear factor-erythroid-2-related factor 2 (Nrf2) helps protect the kidney against oxidative stress by playing a pivotal role in the cooperative induction of genes that encode antioxidant and detoxifying enzymes. Nrf2 is confined to the cytoplasm as an inactive complex bound to a repressor Kelch-like ECH-associated protein 1 (Keap1), which facilitates ubiquitination of Nrf2. Studies using CKD model animals showed that despite stimulated oxidative stress the nuclear Nrf2 level was suppressed, which led to downregulation of the antioxidant enzymes. Hence, deterioration in Nrf2-Keap1 signaling could contribute to the severity of oxidative stress and the progression of CKD. By contrast, acute kidney injury (AKI) induces activation of renal Nrf2. Nrf2 activators or its proteasomal degradation inhibitors enhance nuclear Nrf2 translocation, inducing potential renoprotective actions against CKD and AKI. In both chronic and acute kidney diseases, sulfate-conjugated uremic toxins appear to enhance ROS production when accumulated in renal cells. An intestinal indole adsorbent ameliorates the progression of CKD by decreasing accumulation of indoxyl sulfate. Therapeutic approaches to prevent oxidative stress via activation of the Nrf2-Keap1 signaling and/or suppression of uremic toxin-induced ROS production could be effective strategies for maintaining kidney function.

Keywords: Abbreviations; ADMA; asymmetric dimethylarginine; AGE; advanced glycation end product; AKI; acute kidney injury; CKD; chronic kidney disease; CVD; cardiovascular disease; ERK; extracellular signal-regulated kinase; GLC; glutamate cysteine ligase; GSH; glutathione; GST; GSH S-transferase; HO-1; heme oxygenase-1; JNK; c-Jun N-terminal kinase; Keap1; Kelch-like ECH-associated protein 1; MCP-1; monocyte chemoattractant protein-1; NADPH; nicotinamide adenine dinucleotide phosphate; NO; nitric oxide; NF-κB; nuclear factor-κB; NQO1; NADPH quinone oxidoreductase 1; Nrf2; nuclear factor erythroid 2-related factor 2; PPAR-γ; peroxisome proliferator-activated receptor-γ ROSreactive oxygen species; SOD; superoxide dismutaseNrf2; Chronic kidney disease; Acute kidney injury; Uremic toxins; Renal tubules

IκB kinase ɛ (IKKɛ): A therapeutic target in inflammation and cancer by Kelly Verhelst; Lynn Verstrepen; Isabelle Carpentier; Rudi Beyaert (pp. 873-880).

The innate immune system forms our first line of defense against invading pathogens and relies for a major part on the activation of two transcription factors, NF-κB and IRF3. Signaling pathways that activate these transcription factors are intertwined at the level of the canonical IκB kinases (IKKα, IKKβ) and non-canonical IKK-related kinases (IKKɛ, TBK1). Recently, significant progress has been made in understanding the function and mechanism of action of IKKɛ in immune signaling. In addition, IKKɛ impacts on cell proliferation and transformation, and is thereby also classified as an oncogene. Studies with IKKɛ knockout mice have illustrated a key role for IKKɛ in inflammatory and metabolic diseases. In this review we will highlight the mechanisms by which IKKɛ impacts on signaling pathways involved in disease development and discuss its potential as a novel therapeutic target.

Keywords: IKKɛ; Inflammation; Cancer; NF-κB; IRF3

Design, synthesis, inhibition studies, and molecular modeling of pepstatin analogues addressing different secreted aspartic proteinases of Candida albicans by Cosimo D. Cadicamo; Jérémie Mortier; Gerhard Wolber; Marie Hell; Ina E. Heinrich; Dana Michel; Lydia Semlin; Ursula Berger; Hans C. Korting; Hans-Dieter Höltje; Beate Koksch; Claudia Borelli (pp. 881-887).

Secreted aspartyl proteinases inhibition study of pepstatin A analogues modified in the P3, P2, and P2′ positions.The family of secreted aspartic proteinases is known as an important virulence factor of yeast infections by Candida albicans in particular, which is the most common fungal pathogen for humans with respect to systemic disease. Due to the continuing increase of drug resistant strains, these proteinases are currently considered as promising drug target candidates. Based on the known Sap2-substrate specificity data and X-ray analyses of Sap/inhibitor complexes, three libraries of inhibitors were designed and synthesized by modifying the structure of pepstatin A, a common non-selective aspartic proteinase inhibitor, at the P3, P2, or P2′ position. These novel inhibitors showed high inhibitory potencies for the isoenzymes Sap1, Sap3, Sap5 and Sap6. Then, the affinity and selectivity of the peptide ligands were investigated by molecular modeling, highlighting new key structural information for the design of potent and selective anti-virulence agents targeting Candida albicans.

Keywords: Abbreviations; Abu; amino-butyric acid; ACN; acetonitrile; Boc; tert; -butyloxycarbonyl; CI; 95% Confidence Interval; Dab; diaminobutyric acid; DBU; 1,8-diazabicyclo[5.4.0]undec-7-ene; DCM; dichloromethane; DIC; N; ,; N; -diisopropylcarbodiimide; DIPEA; N; ,; N; -diisopropylethylamine; DMF; dimethylformamide; ESI; electrospray ionization; Fmoc; 9-fluorenylmethoxycarbonyl; HOAt; 1-hydroxy-7-azabenzotriazole; HPLC; high performance liquid chromatography; Nle; nor; -leucine; O; -MeTyr; O; -methyl-tyrosine; Orn; ornithine; Pbf; 2,2,4,6,7-pentamethyldihydro-benzo-furan-5-sulfonyl; p; -MePhe; para-methyl-phenylalanine; TBS; tert; -butyldimethylsilyl; TFA; trifluoroacetic acid; TIS; triisopropylsilane; Sap; secreted aspartic proteinases; Sta; statine; SPPS; solid phase peptide synthesisAnti-virulence agent; Peptidomimetic; Candidapeptidase; Secreted aspartic proteinases inhibition; Pepstatin; Candida albicans

Inhibition of T-type calcium channels disrupts Akt signaling and promotes apoptosis in glioblastoma cells by Nicholas C.K. Valerie; Barbara Dziegielewska; Amol S. Hosing; Ewa Augustin; Lloyd S. Gray; David L. Brautigan; James M. Larner; Jaroslaw Dziegielewski (pp. 888-897).

Glioblastoma multiforme (GBM) are brain tumors that are exceptionally resistant to both radio- and chemotherapy regimens and novel approaches to treatment are needed. T-type calcium channels are one type of low voltage-gated channel (LVCC) involved in embryonic cell proliferation and differentiation; however they are often over-expressed in tumors, including GBM. In this study, we found that inhibition of T-type Ca²⁺ channels in GBM cells significantly reduced their survival and resistance to therapy. Moreover, either T-type selective antagonists, such as mibefradil, or siRNA-mediated knockdown of the T-type channel alpha subunits not only reduced cell viability and clonogenic potential, but also induced apoptosis. In response to channel blockade or ablation, we observed reduced phosphorylation of Akt and Rictor, suggesting inhibition of the mTORC2/Akt pathway. This was followed by reduction in phosphorylation of anti-apoptotic Bad and caspases activation. The apoptotic response was specific for T-type Ca²⁺ channels, as inhibition of L-type Ca²⁺ channels did not induce similar effects. Our results implicate T-type Ca²⁺ channels as distinct entities for survival signaling in GBM cells and suggest that they are a novel molecular target for tumor therapy.

Keywords: Abbreviations; CACNA1C; L-type channel subunit Cav1.2; CACNA1G; T-type channel subunit Cav3.1; CACNA1H; T-type channel subunit Cav3.2; GBM; glioblastoma multiforme; LVCC; low voltage gated calcium channelT-type calcium channels; Apoptosis; Glioblastoma multiforme; Akt; mTORC2; Mibefradil

Morin inhibits STAT3 tyrosine 705 phosphorylation in tumor cells through activation of protein tyrosine phosphatase SHP1 by Subash C. Gupta; Kanokkarn Phromnoi; Bharat B. Aggarwal (pp. 898-912).

The major goal of cancer drug discovery is to find an agent that is safe and affordable, yet effective against cancer. Here we show that morin (3,5,7,2′,4′-pentahydroxyflavone) has potential against cancer cells through suppression of the signal transducer and activator of transcription 3 (STAT3) pathway, which is closely linked to the transformation, survival, proliferation, and metastasis of cancer. We found that morin completely suppressed inducible and constitutively activated STAT3 and blocked the nuclear translocation of STAT3 and its DNA binding in multiple myeloma and head and neck squamous carcinoma cells. Morin inhibited activated Src, JAK-1, and JAK-2, all of which are linked to STAT3 activation, while up-regulating a protein inhibitor of activated STAT3, PIAS3. Pervanadate reversed the effects of morin on STAT3 phosphorylation, indicating the role of a protein tyrosine phosphatase. Furthermore, morin induced SHP1 expression at both the mRNA and protein levels, and silencing of SHP1 abrogated the effect of morin on STAT3 phosphorylation, indicating that morin mediates its effects on STAT3 through SHP1. Suppression of STAT3 correlated with the down-regulation of various gene products linked to tumor survival, proliferation, and angiogenesis and led to sensitization of tumor cells to thalidomide and bortezomib. Comparing the activities of morin with those of four structurally related flavonols demonstrated the importance of hydroxyl groups in the B ring in inhibiting STAT3 activation. These findings suggest that morin suppresses the STAT3 pathway, leading to the down-regulation of STAT3-dependent gene expression and chemosensitization of tumor cells.

Keywords: Apoptosis; Chemosensitization; Flavonol; SHP-1; STAT3

Saxifragifolin D induces the interplay between apoptosis and autophagy in breast cancer cells through ROS-dependent endoplasmic reticulum stress by Jun-Min Shi; Liang-Liang Bai; Dong-Mei Zhang; Anita Yiu; Zhi-Qi Yin; Wei-Li Han; Jun-Shan Liu; Yong Li; Deng-Yue Fu; Wen-Cai Ye (pp. 913-926).

Breast cancer is the leading cause of cancer death among females, and novel chemotherapeutic drugs for treating breast cancer are needed urgently. Saxifragifolin D (SD) was isolated by our group from Androsace umbellata which is commonly used to treat solid tumor. In this study, we evaluated its growth inhibitory effect on breast cancer cells and explored the underlying molecular mechanisms. Our results showed that SD inhibited the growth of both MCF-7 and MDA-MB-231 cells significantly. Mechanistic studies demonstrated that SD induced apoptosis through mitochondrial apoptotic pathway. Evidence of SD-induced autophagy included the occurrence of autophagic vacuoles, up-regulation of LC3-II, Beclin1 and Vps34. Inhibition of autophagy by bafilomycin A1 or Beclin1 siRNA pretreatment decreased the ratio of apoptosis, indicating that autophagy induction contributes to apoptosis and is required for the latter. SD was also found to induce endoplasmic reticulum stress, accompanied by ROS production, increase of intracellular calcium and up-regulation of Bip, IRE1α and XBP-1s. Inhibition of endoplasmic reticulum stress by N-acetyl-l-cysteine, tauroursodeoxycholic acid or IRE1α siRNA pretreatment could suppress both apoptosis and autophagy. Besides, increases in CHOP, calnexin, calpain, p-JNK and p-Bcl-2 were followed by subsequent dissociation of Beclin1 from Bcl-2, further suggesting endoplasmic reticulum stress to be the common signaling pathway shared by SD-induced apoptosis and autophagy. In conclusion, SD inhibits breast cancer cell growth and induces interplay between apoptosis and autophagy through ROS-mediated endoplasmic reticulum stress. It will provide molecular bases for developing SD into a drug candidate for the treatment of breast cancer.

Keywords: Saxifragifolin D; Breast cancer; Autophagy; Apoptosis; Endoplasmic reticulum stress

ABT-737 resistance in B-cells isolated from chronic lymphocytic leukemia patients and leukemia cell lines is overcome by the pleiotropic kinase inhibitor quercetin through Mcl-1 down-regulation by Maria Russo; Carmela Spagnuolo; Silvestro Volpe; Idolo Tedesco; Stefania Bilotto; Gian Luigi Russo (pp. 927-936).

Chronic lymphocytic leukemia (CLL) is the most frequent form of leukemia in adult population and despite numerous studies, it is considered an incurable disease. Since CLL is characterized by overexpression of pro-survival Bcl-2 family members, treatments with their antagonists, such as ABT-737, represent a promising new therapeutic strategy. ABT-737 is a BH3 mimetic agent which binds Bcl-2, Bcl-X_L and Bcl-w with high affinity, while weakly interacts with Mcl-1 and Bfl-1. Previous studies demonstrated that quercetin, a flavonoid naturally present in food and beverages, was able to sensitize B-cells isolated from CLL patients to apoptosis when associated with death ligands or fludarabine, through a mechanism involving Mcl-1 down-regulation. Here, we report that the association between ABT-737 and quercetin synergistically induces apoptosis in B-cells and in five leukemic cell lines (Combination Index <1). Peripheral blood mononuclear cell from healthy donors were not affected by quercetin treatment. The molecular pathways triggered by quercetin have been investigated in HPB-ALL cells, characterized by the highest resistance to both ABT-737 and quercetin when applied as single molecules, but highly sensitivity to the co-treatment. In this cell line, quercetin down-regulated Mcl-1 through the inhibition of PI₃K/Akt signaling pathway, leading to Mcl-1 instability. The same mechanism was confirmed in B-cells. These results may open new clinical perspectives based on a translational approach in CLL therapy.

Keywords: Abbreviations; Bcl-2; B-cell lymphoma; BH3; Bcl-2 homology domain 3; CI; combination index; CLL; chronic lymphocytic leukemia; CR; complete remission; Mcl-1; myeloid cell leukemia 1; MEK; mitogen-activated protein kinase kinase; PI; ₃; K; phosphatidylinositol-3-kinase; PBMC; peripheral blood mononuclear cell; PS; phosphatidylserine; qPCR; quantitative PCRABT-737; Quercetin; Mcl-1; Chronic lymphocytic leukemia; Apoptosis

l-Carnitine protects against arterial hypertension-related cardiac fibrosis through modulation of PPAR-γ expression by Sonia Zambrano; Antonio J. Blanca; María V. Ruiz-Armenta; José L. Miguel-Carrasco; Miguel Arévalo; María J. Vázquez; Alfonso Mate; Carmen M. Vázquez (pp. 937-944).

Cardiac fibrosis is a pathogenic factor in a variety of cardiovascular diseases and is characterized by an abnormal accumulation of extracellular matrix protein that leads to cardiac dysfunction.l-Carnitine (LC) plays an essential role in the β-oxidation of long-chain fatty acids in lipid metabolism. We have previously demonstrated the beneficial effects of LC in hypertensive rats. The aim of this study was to analyze the effect of LC on arterial hypertension-associated cardiac fibrosis and to explore the mechanisms of LC action. To this end, four groups of rats were used: Wistar (control), rats treated with 400mg/kg/day of LC, rats treated with 25mg/kg/day ofl-NAME (to induce hypertension), and rats treated with LC+l-NAME simultaneously. We found an elevation in the myocardial expression of profibrotic factors (TGF-β₁ and CTGF), types I and III of collagen, and NADPH oxidase subunits (NOX2 and NOX4), in hypertensive rats when compared with normotensive ones. In addition, an increase in myocardial fibrosis was also found in thel-NAME group. These results were accompanied by a down-regulation of PPAR-γ in the heart of hypertensive animals. When hypertensive rats were treated with LC, all these alterations were reversed. Moreover, a significant negative correlation was observed between myocardial interstitial fibrosis and mRNA expression of PPAR-γ. In conclusion, the reduction of cardiac fibrosis and the down-regulation of NOX2, NOX4, TGF-β₁ and CTGF induced by LC might be, at least in part, mediated by an upregulation of PPAR-γ, which leads to a reduction on hypertension-related cardiac fibrosis.

Keywords: l; -Carnitine; Fibrosis; PPAR; Heart; l; -NAME; Hypertension

Anti-human VWF monoclonal antibody SZ-123 prevents arterial thrombus formation by inhibiting VWF–collagen and VWF–platelet interactions in Rhesus monkeys by Yi-ming Zhao; Miao Jiang; Shun-dong Ji; Yang He; Fei Shen; Xue-mei Li; Chang-geng Ruan (pp. 945-953).

(A) Closed conformation of A1-A2-A3. (B) Unfolded conformation of A1-A2-A3 induced by high shear. (C). SZ-123 inhibits interaction between VWF A3 and collagen, and also induces binding of A3 to A1.The interactions between collagen, von Willebrand factor (VWF), and glycoprotein Ib (GPIb) are crucial for hemostasis and thrombosis. This axis represents a promising target for the development of new antithrombotic agents. In this study, we investigate the in vivo antithrombotic efficacy of an anti-VWF monoclonal antibody SZ-123 and its potential underlying mechanisms. Cyclic flow reductions (CFRs), an indicator of arterial thrombosis, were measured in the femoral artery of anesthetized Rhesus monkeys before and after intravenous administration of SZ-123. Ex vivo VWF binding to collagen, platelet agglutination, platelet count, and template bleeding time were used as measurements of antithrombotic activity. In addition, plasma VWF and SZ-123 levels, and VWF occupancy were measured by ELISA. Administration of 0.1, 0.3, and 0.6mg/kg SZ-123 resulted in 45.3%, 78.2%, and 100% reductions in CFRs, respectively. When 0.3 and 0.6mg/kg SZ-123 were administered, 100% of VWF was occupied by the antibody. Moreover, 100% ex vivo inhibition of VWF–collagen binding and 60–95% inhibition of platelet agglutination were observed from 15min to 1h. None of the doses resulted in significant prolongation of bleeding time. In vitro experiments revealed that SZ-123 not only blocks the collagen–VWF A3 interaction but also indirectly inhibits VWF A1 binding to GPIbα induced by ristocetin. Thus, we demonstrate that SZ-123 prevents in vivo arterial thrombus formation under high shear conditions by inhibiting VWF A3–collagen and VWF A1–platelet interactions and does not significantly prolong bleeding time.

Keywords: Abbreviations; VWF; von Willebrand factor (VWF); GPIbα; glycoprotein Ibα; CFRs; cyclic flow reductionsThrombosis; Collagen; VWF; Platelet; Platelet adhesion; SZ-123

ATP stimulates PGE₂/cyclin D1-dependent VSMCs proliferation via STAT3 activation: Role of PKCs-dependent NADPH oxidase/ROS generation by I-Ta Lee; Chih-Chung Lin; Chao-Hung Wang; Wen-Jin Cherng; Jong-Shyan Wang; Chuen-Mao Yang (pp. 954-964).

Vascular smooth muscle cells (VSMCs) that function as synthetic units play important roles in cardiovascular diseases. Extracellular nucleotides, such as ATP, have been shown to act via activation of P₂ purinoceptors implicated in various inflammatory diseases, we hypothesized that extracellular nucleotides contribute to vascular diseases via up-regulation of inflammatory proteins, including cyclooxygenase-2 (COX-2) and cytosolic phospholipase A₂ (cPLA₂) in VSMCs. However, the mechanisms of ATP-induced cPLA₂ and COX-2 expression and PGE₂ synthesis remain largely unclear. We showed that pretreatment with the inhibitors of STAT3 (CBE), NADPH oxidase [diphenyleneiodonium chloride (DPI) or apocynin (APO)], ROS [N-acetyl-l-cysteine (NAC)], and PKC (Ro-318220, Gö6983, or Rottlerin) or transfection with siRNAs of STAT3 and p47^phox markedly inhibited ATPγS-induced cPLA₂ and COX-2 mRNA/protein expression and promoter activity and PGE₂ secretion. ATPγS further stimulated PKC, p47^phox, and STAT3 translocation. Moreover, ATPγS-induced STAT3 phosphorylation and translocation was inhibited by pretreatment with the inhibitors of PKC, NADPH oxidase, and ROS. ATPγS enhanced NADPH oxidase activity and ROS generation in VSMCs, which were reduced by pretreatment with Ro-318220, Gö6983, or Rottlerin. Finally, we found that ATPγS significantly induced cyclin D1 expression and VSMCs proliferation, which were inhibited by pretreatment with NAC, APO, DPI, Ro-318220, Gö6983, Rottlerin, or CBE or transfection with siRNAs of COX-2 and cyclin D1. We also demonstrated that ATPγS induced cyclin D1 expression via a PGE₂-dependent pathway. These results suggested that ATPγS-induced cPLA₂/COX-2 expression and PGE₂ secretion is mediated through a PKC/NADPH oxidase/ROS/STAT3-dependent pathway in VSMCs.

Keywords: Cyclin D1; Cyclooxygenase-2; Cytosolic phospholipase A; ₂; NADPH oxidase

Anti-obesity effects of 3-hydroxychromone derivative, a novel small-molecule inhibitor of glycogen synthase kinase-3 by Sooho Lee; Woo Kyeom Yang; Ji Ho Song; Young Min Ra; Jin-Hyun Jeong; Wonchae Choe; Insug Kang; Sung-Soo Kim; Joohun Ha (pp. 965-976).

Glycogen synthase kinase 3 (GSK-3) plays a central role in cellular energy metabolism, and dysregulation of GSK-3 activity is implicated in a variety of metabolic disorders, including obesity, type 2 diabetes, and cancer. Hence, GSK-3 has emerged as an attractive target molecule for the treatment of metabolic disorders. Therefore, this research focused on identification and characterization of a novel small-molecule GSK-3 inhibitor. Compound1a, a structure based on 3-hydroxychromone bearing isothiazolidine-1,1-dione, was identified from chemical library as a highly potent GSK-3 inhibitor. An in vitro kinase assay utilizing a panel of kinases demonstrated that compound1a strongly inhibits GSK-3β. The potential effects of compound1a on the inactivation of GSK-3 were confirmed in human liver HepG2 and human embryonic kidney HEK293 cells. Stabilization of glycogen synthase and β-catenin, which are direct targets of GSK-3, by compound1a was assessed in comparison with two other GSK-3 inhibitors: LiCl and SB-415286. In mouse 3T3-L1 preadipocytes, compound1a markedly blocked adipocyte differentiation. Consistently, intraperitoneal administration of compound1a to diet-induced obese mice significantly ameliorated their key symptoms such as body weight gain, increased adiposity, dyslipidemia, and hepatic steatosis due to the marked reduction of whole-body lipid level. In vitro and in vivo effects were accompanied by upregulation of β-catenin stability and downregulation of the expression of several critical genes related to lipid metabolism. From these results, it can be concluded that compound1a, a novel small-molecule inhibitor of GSK-3, has potential as a new class of therapeutic agent for obesity treatment.

Keywords: Abbreviations; GSK-3; glycogen synthase kinase 3; GS; glycogen synthase; NFD; normal fat diet; HFD; high-fat diet; DIO; diet-induced obesity; PEG400; polyethylene glycol 400; HRP; horseradish peroxidase; DMEM; Dulbecco's modified Eagle's medium; FBS; fetal bovine serum; CS; calf serum; PPARγ; peroxisome proliferator-activated receptor γ; C/EBPα; CCAAT/enhancer-binding protein α; FAS; fatty acid synthase; SREBP1c; sterol regulatory element-binding protein 1c; aP2; adipocyte fatty acid binding protein; 11β-HSD1; 11β-hydroxysteroid dehydrogenase 1; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; TBS; Tris-buffered saline; H–E; hematoxylin–eosin; GTT; glucose tolerance test; p90rsk2; p90 ribosomal protein S6 kinase 2; WAT; white adipose tissue; TCF/LEF; T-cell factor/lymphoid-enhancing factorObesity; Lipid metabolism; Protein kinase inhibitor; GSK-3

Thioredoxin-mimetic peptides (TXM) reverse auranofin induced apoptosis and restore insulin secretion in insulinoma cells by Moshe Cohen-Kutner; Lena Khomsky; Michael Trus; Yonatan Aisner; Masha Y. Niv; Moran Benhar; Daphne Atlas (pp. 977-990).

The thioredoxin reductase/thioredoxin system (TrxR/Trx1) plays a major role in protecting cells from oxidative stress. Disruption of the TrxR-Trx1 system keeps Trx1 in the oxidized state leading to cell death through activation of the ASK1-Trx1 apoptotic pathway. The potential mechanism and ability of tri- and tetra-oligopeptides derived from the canonical -CxxC- motif of the Trx1-active site to mimic and enhance Trx1 cellular activity was examined. The Trx mimetics peptides (TXM) protected insulinoma INS 832/13 cells from oxidative stress induced by selectively inhibiting TrxR with auranofin (AuF). TXM reversed the AuF-effects preventing apoptosis, and increasing cell-viability. The TXM peptides were effective in inhibiting AuF-induced MAPK, JNK and p38^MAPK phosphorylation, in correlation with preventing caspase-3 cleavage and thereby PARP-1 dissociation. The ability to form a disulfide-bridge-like conformation was estimated from molecular dynamics simulations. The TXM peptides restored insulin secretion and displayed Trx1 denitrosylase activity. Their potency was 10–100-fold higher than redox reagents like NAC, AD4, or ascorbic acid. Unable to reverse ERK1/2 phosphorylation, TXM-CB3 (NAc-Cys-Pro-Cys amide) appeared to function in part, through inhibiting ASK1-Trx dissociation. These highly effective anti-apoptotic effects of Trx1 mimetic peptides exhibited in INS 832/13 cells could become valuable in treating adverse oxidative-stress related disorders such as diabetes.

Keywords: Thioredoxin; Oxidative stress; Diabetes; Insulin secretion; MAPK; Apoptosis; Molecular dynamics; ASK1

AMP-activated protein kinase as regulator of P2Y₆ receptor-induced insulin secretion in mouse pancreatic β-cells by Ramachandran Balasubramanian; Hiroshi Maruoka; P. Suresh Jayasekara; Zhan-Guo Gao; Kenneth A. Jacobson (pp. 991-998).

5′-AMP-activated protein kinase (AMPK) and its pharmacological modulators have been targeted for treating type 2 diabetes. Extracellular uridine 5′-diphosphate (UDP) activates P2Y₆ receptors (P2Y₆Rs) in pancreatic β-cells to release insulin and reduce apoptosis, which would benefit diabetes. Here, we studied the role of P2Y₆R in activation of AMPK in MIN6 mouse pancreatic β-cells and insulin secretion. Treatment with a potent P2Y₆R dinucleotide agonist MRS2957 (500nM) activated AMPK, which was blocked by P2Y₆R-selective antagonist MRS2578. Also, MRS2957 induced phosphorylation of acetyl-coenzyme A carboxylase (ACC), a marker of AMPK activity. Calcium chelator BAPTA-AM, calmodulin-dependent protein kinase kinase (CaMKK) inhibitor STO-069 and IP₃ receptor antagonist 2-APB attenuated P2Y₆R-mediated AMPK phosphorylation revealing involvement of intracellular Ca²⁺ pathways. P2Y₆R agonist induced insulin secretion at high glucose, which was reduced by AMPK siRNA. Thus, P2Y₆R has a crucial role in β-cell function, suggesting its potential as a therapeutic target in diabetes.

Keywords: Abbreviations; AMPK; AMP-activated protein kinase; BAPTA-AM; 1,2-; bis; (2-aminophenoxy)ethane-; N; ,; N; ,; N; ′,; N; ′-tetraacetic acid tetrakis(acetoxymethyl ester); BIM; bisindolylmaleimide I; CaMKK; Ca; ²⁺; /calmodulin-dependent protein kinase kinase; DMEM; Dulbecco's modified Eagle's medium; ECL; enhanced chemiluminescence; FBS; fetal bovine serum; GSIS; glucose-stimulated insulin secretion; HRP; horseradish peroxidase; IP; ₃; inositol trisphosphate; KRBB; Kreb's ringer bicarbonate buffer; LKB1; liver kinase B1; MRS2693; 5-iodouridine 5′-diphosphate; MRS2957; P; ¹; -(uridine 5′-)-P; ⁴; -(; N; ⁴; -methoxycytidine 5′-)tetraphosphate; MRS2578; N; ,; N; ″-1,4-butanediyl-; bis; [; N; ′-(3-isothiocyanatophenyl)thiourea; PI3K; phosphatidylinositol 3-kinase; PKC; protein kinase C; PLC; phospholipase C; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; STO-609; 7-oxo-7; H; -benzimidazo[2,1-; a; ]benz[de]isoquinoline-3-carboxylic acid acetate; TNF-α; tumor necrosis factor-α; UDP; uridine 5′-diphosphate; Tris; Tris(hydroxymethyl)aminomethaneNucleotides; G protein-coupled receptor; Insulin; AMPK; Diabetes; P2Y; ₆; receptor

p63 involvement in poly(ADP-ribose) polymerase 1 signaling of topoisomerase I-dependent DNA damage in carcinoma cells by Daniela Montariello; Annaelena Troiano; Maria Malanga; Viola Calabrò; Piera Quesada (pp. 999-1006).

Poly(ADP-ribose)polymerase 1 (PARP-1) inhibitors are thought as breakthrough for cancer treatment in solid tumors such as breast cancer through their effects on PARP's enzymatic activity. Our previous findings showed that the hydrophilic PARP inhibitor PJ34 enhances the sensitivity of p53 proficient MCF7 breast carcinoma cells to topotecan, a DNA Topoisomerase I (TOP 1) inhibitor.In the present study, we combine the classical TOP 1 poison camptothecin or its water-soluble derivative topotecan with PJ34 to investigate the potentiation of chemotherapeutic efficiency in MCF7 (p53^WT), MDA-MB231 (p53^mut) breast carcinoma cells and SCC022 (p53^null) squamous carcinoma cells.We show that, following TPT-PJ34 combined treatment, MCF7 cells exhibit apoptotic death while MDA-MB231 and SCC022 cells are more resistant to these agents. Specifically, in MCF7, (i) PJ34 in combination with TPT causes a G2/M cell cycle arrest followed by massive apoptosis; (ii) PJ34 addition reverts TPT-dependent PARP-1 automodification and triggers caspase-dependent PARP-1 proteolysis; (iii) TPT, used as a single agent, stimulates p53 expression while in combination with PJ34 increases p53, TAp63α and TAp63γ protein levels with a concomitant reduction of MDM2 protein.The identification of p63 proteins as new players involved in the cancer cell response to TPT-PJ34 is relevant for a better understanding of the PARP1-dependent signaling of DNA damage. Furthermore, our data indicate that, in response to TPT-PJ34 combined chemotherapy, a functional cooperation between p53 and TAp63 proteins may occur and be essential to trigger apoptotic cell death.

Keywords: TOP I inhibitors; PARP1 inhibitors; P53; P63; Carcinoma cells

Transcriptome profiling and genome-wide DNA binding define the differential role of fenretinide and all-trans RA in regulating the death and survival of human hepatocellular carcinoma Huh7 cells by Ying Hu; Hui-Xin Liu; Yuqi He; Yaping Fang; Jianwen Fang; Yu-Jui Yvonne Wan (pp. 1007-1017).

Fenretinide is significantly more effective in inducing apoptosis in cancer cells than all-trans retinoic acid (ATRA). The current study uses a genome-wide approach to understand the differential role fenretinide and ATRA have in inducing apoptosis in Huh7 cells. Fenretinide and ATRA-induced gene expressions and DNA bindings were profiled using microarray and chromatin immunoprecipitation with anti-RXRα antibody. The data showed that fenretinide was not a strong transcription regulator. Fenretinide only changed the expressions of 1 093 genes, approximately three times less than the number of genes regulated by ATRA (2 811). Biological function annotation demonstrated that both fenretinide and ATRA participated in pathways that determine cell fate and metabolic processes. However, fenretinide specifically induced Fas/TNFα-mediated apoptosis by increasing the expression of pro-apoptotic genes i.e., DEDD2, CASP8, CASP4, and HSPA1A/B; whereas, ATRA induced the expression of BIRC3 and TNFAIP3, which inhibit apoptosis by interacting with TRAF2. In addition, fenretinide inhibited the expression of the genes involved in RAS/RAF/ERK-mediated survival pathway. In contrast, ATRA increased the expression of SOSC2, BRAF, MEK, and ERK genes. Most genes regulated by fenretinide and ATRA were bound by RXRα, suggesting a direct effect. This study revealed that by regulating fewer genes, the effects of fenretinide become more specific and thus has fewer side effects than ATRA. The data also suggested that fenretinide induces apoptosis via death receptor effector and by inhibiting the RAS/RAF/ERK pathway. It provides insight on how retinoid efficacy can be improved and how side effects in cancer therapy can be reduced.

Keywords: Abbreviations; APL; acute promyelocytic leukemia; ATRA; all-trans retinoic acid; ChIP-Seq; chromatin immunoprecipitation followed by next generation sequencing; DAVID; the Database for Annotation, Visualization and Integrated Discovery; DR; direct repeat; ER; everted repeat; ER stress; endoplasmic reticulum stress; ERK; extracellular-signal-regulated kinase; FDR; false discovery ratio; IR; inverted repeat; RA; retinoic acid; RAR; retinoic acid receptor; ROS; reactive oxygen species; RXR; retinoid X receptor; qRT-PCR; real-time quantitative reverse transcription PCR; qPCR; quantitative PCR; TNF; tumor necrosis factorRetinoic acid receptor; Retinoid X receptor; Nuclear receptor; Hepatocellular carcinoma; ChIP-Seq

Osteoblasts survive the arsenic trioxide treatment by activation of ATM-mediated pathway by Yu-Chen Hu; Bau-Shan Hsieh; Hsiao-Ling Cheng; Li-Wen Huang; Tzu-Ching Huang; I-Yu Huang; Kee-Lung Chang (pp. 1018-1026).

Arsenic trioxide (ATO) is widely used in tumor treatment, but excessive arsenic exposure can have adverse effects. We recently found that, in primary osteoblasts, ATO produces oxidative stress and causes DNA tailing, but does not induce apoptosis. We further examined the signaling pathway by which osteoblasts survive ATO treatment, and found that they were arrested at G2/M phase of the cell cycle at 30h and overrode the G2/M boundary at 48h. After treatment for 30h, there was increased Cdc2 phosphorylation and expression of Wee1, a Cdc2 kinase, and expression of the cell cycle inhibitor, p21^waf1/cip1, which interacts with Cdc2. Furthermore, levels of the phosphatase Cdc25C, which activates Cdc2, were decreased, while the ratio of its phosphorylated/inactivated form to the total amount was increased. Moreover, phosphorylation/activation of the checkpoint kinases Chk1, Chk2 and p53 levels were increased, as were levels of activated ATM and γ-H2AX. The cell viability was decreased as an ATM inhibitor was added. Additionally, these effects of ATO on γ-H2AX, Chk1, Chk2, p53, and p21^waf1/cip1 were reduced by an ATM inhibitor. These findings suggest that G2/M phase arrest of osteoblasts is mediated by Chk1/Chk2 activation via an ATM-dependent pathway by which osteoblasts survive.

Keywords: Abbreviations; ATO; arsenic trioxide; ROS; reactive oxygen species; Cdks; cyclin-dependent kinases; PI3-Ks; phosphoinositide 3-kinase related kinases; ATM; ataxia telangiectasia-mutated; ATR; ataxia telangiectasia-mutated and Rad3-related; ATRIP; ATR interacting protein; DNA-PK; DNA-dependent protein kinase; Rb; retinoblastoma protein; MEM; minimal essential medium; FBS; fetal bovine serum; IACUC; Institutional Animal Care and Use Committee; APL; acute promyelocytic leukemia; OB; primary osteoblastOsteoblast; Arsenic trioxide; Checkpoint kinase; ATM; Cell cycle

Sections

Personal tools

Biochemical Pharmacology (v.85, #7)

Sections

Personal tools

Local resources

Biochemical Pharmacology (v.85, #7)