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Biochemical Pharmacology (v.81, #11)

Editorial Advisory Board (pp. iii).

Suppression of Stat3 activity sensitizes gefitinib-resistant non small cell lung cancer cells by Huan-Chih Chiu; Ding-Li Chou; Chin-Ting Huang; Wen-Hsing Lin; Tzu-Wen Lien; Kuei-Jung Yen; John T.-A. Hsu (pp. 1263-1270).

Epidermal growth factor receptor (EGFR) is a proven therapeutic target to treat a small subset of non small cell lung cancer (NSCLC) harboring activating mutations within the EGFR gene. However, many NSCLC patients are not sensitive to EGFR inhibitors, suggesting that other factors are implicated in survival of NSCLC cells. Signal transducers and activators of transcription 3 (Stat3) function as transcription factor to mediate cell survival and differentiation and the dysregulation of Stat3 has been discovered in a number of cancers. In this study, we found that a small molecule, reactivation of p53 and induction of tumor cell apoptosis (RITA), showed anti-cancer activity against gefitinib-resistant H1650 cells through a p53-independent pathway. Stat3 suppression by RITA attracted our attention to investigate the role of Stat3 in sustaining survival of H1650 cells. Pharmacological and genetic approaches were employed to down-regulate Stat3 in H1650 cells. WP1066, a known Stat3 inhibitor, was shown to exhibit inhibitory effect on the growth of H1650 cells. Meanwhile, apoptosis activation by siRNA-mediated down-regulation of Stat3 in H1650 cells provides more direct evidence for the involvement of Stat3 in viability maintenance of H1650 cells. Moreover, as a novel identified Stat3 inhibitor, RITA increased doxorubicin sensitivity of H1650 cells in vitro and in vivo, suggesting that doxorubicin accompanied with Stat3 inhibitors may be considered as an alternative strategy to treat NSCLC patients who have inherent resistance to doxorubicin. Overall, our observations reveal that targeting Stat3 may be an effective treatment for certain NSCLC cells with oncogenic addition to Stat3.

Keywords: RITA; Stat3; WP1066; Doxorubicin sensitivity; Apoptosis

The signalling axis mediating neuronal apoptosis in response to [Pt(O,O′-acac)(γ-acac)(DMS)] by Antonella Muscella; Nadia Calabriso; Carla Vetrugno; Francesco Paolo Fanizzi; Sandra Angelica De Pascali; Santo Marsigliante (pp. 1271-1285).

It was previously shown that [Pt(O,O′-acac)(γ-acac)(DMS)] induces apoptosis in various cancer cells and exerts antimetastatic responses in vitro. In rats, [Pt(O,O′-acac)(γ-acac)(DMS)] reaches the central nervous system in quantities higher than cisplatin causing less excitotoxicity. The aim of the present paper was to investigate whether [Pt(O,O′-acac)(γ-acac)(DMS)] is able to exert cytotoxic effects on SH-SY5Y human neuroblastoma cell line, and to study the intracellular transduction mechanisms underlying these effects. Here we have demonstrated that [Pt(O,O′-acac)(γ-acac)(DMS)] was more effective than cisplatin in provoking apoptosis characterized by: (a) mitochondria depolarization, (b) decrease of Bcl-2 expression and increase of BAX expressions with cytosol-to-mitochondria translocation, (c) activation of caspase-7 and -9 and (d) generation of reactive oxygen species (ROS). [Pt(O,O′-acac)(γ-acac)(DMS)] provoked the activation of the following signalling kinases that were interacting with each other: PKC-δ and -ɛ, ERK1/2, p38MAPK, JNK1/2, NF-κB, c-src and FAK. We found that ROS generated by NADPH oxidase was responsible for the [Pt(O,O′-acac)(γ-acac)(DMS)]-mediated PKC-δ and -ɛ activation and consequential phosphorylation of all MAPKs. [Pt(O,O′-acac)(γ-acac)(DMS)]-induced mitochondrial apoptosis was blocked when p38MAPK and JNK1/2 were inhibited, whilst the effects on Bax/Bcl-2 mRNA and protein levels were blocked inhibiting NF-κB. NF-κB nuclear translocation was blocked inhibiting MEK1/2 activity. In addition to the induction of apoptosis [Pt(O,O′-acac)(γ-acac)(DMS)] downregulated pro-survival pathway. Survival inhibition started from mitochondrial ROS generation which induced c-src, FAK and Akt activation. In conclusion, our results suggest that [Pt(O,O′-acac)(γ-acac)(DMS)] may be considered a promising compound for the treatment of neuroblastoma. Further studies are warranted to explore in detail the therapeutic potential of this compound.

Keywords: Abbreviations; DMEM; Dulbecco's modification of Eagle's medium; DMS; dimethylsulphide; ECL; enhanced chemiluminescence; NBT; nitroblue tetrazolium; PBS; phosphate-buffered saline; PVDF; polyvinylidene difluoride membrane; ROS; reactive oxygen species; SDS; sodium dodecyl sulphate[Pt(O,O′-acac)(γ-acac)(DMS)]; Apoptosis; PKCs; ROS; SH-SY5Y; MAPKs

WISP-1 increases MMP-2 expression and cell motility in human chondrosarcoma cells by Chun-Han Hou; Yi-Chun Chiang; Yi-Chin Fong; Chih-Hsin Tang (pp. 1286-1295).

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. WISP-1 is a cysteine-rich protein that belongs to the CCN (Cyr61, CTGF, Nov) family of matricellular proteins. However, the effect of WISP-1 on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that WISP-1 increased the migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells (JJ012 cells). We also found that human chondrosarcoma tissues had significant expression of the WISP-1 which was higher than that in normal cartilage. α5β1 monoclonal antibody and MAPK kinase (MEK) inhibitors (PD98059 and U0126) inhibited the WISP-1-induced increase of the migration and MMP-2 up-regulation of chondrosarcoma cells. WISP-1 stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK and extracellular signal-regulated kinase (ERK). In addition, NF-κB inhibitors also suppressed the cell migration and MMP-2 expression enhanced by WISP-1. Moreover, WISP-1 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-2 promoter. Taken together, our results indicated that WISP-1 enhances the migration of chondrosarcoma cells by increasing MMP-2 expression through the α5β1 integrin receptor, FAK, MEK, ERK, p65 and NF-κB signal transduction pathway.

Keywords: WISP-1; Migration; Chondrosarcoma; ERK; NF-κB

The synthetic bryostatin analog Merle 23 dissects distinct mechanisms of bryostatin activity in the LNCaP human prostate cancer cell line by Noemi Kedei; Andrea Telek; Alexandra Czap; Emanuel S. Lubart; Gabriella Czifra; Dazhi Yang; Jinqiu Chen; Tyler Morrison; Paul K. Goldsmith; Langston Lim; Poonam Mannan; Susan H. Garfield; Matthew B. Kraft; Wei Li; Gary E. Keck; Peter M. Blumberg (pp. 1296-1308).

Bryostatin 1 has attracted considerable attention both as a cancer chemotherapeutic agent and for its unique activity. Although it functions, like phorbol esters, as a potent protein kinase C (PKC) activator, it paradoxically antagonizes many phorbol ester responses in cells. Because of its complex structure, little is known of its structure-function relations. Merle 23 is a synthetic derivative, differing from bryostatin 1 at only four positions. However, in U-937 human leukemia cells, Merle 23 behaves like a phorbol ester and not like bryostatin 1. Here, we characterize the behavior of Merle 23 in the human prostate cancer cell line LNCaP. In this system, bryostatin 1 and phorbol ester have contrasting activities, with the phorbol ester but not bryostatin 1 blocking cell proliferation or tumor necrosis factor alpha secretion, among other responses. We show that Merle 23 displays a highly complex pattern of activity in this system. Depending on the specific biological response or mechanistic change, it was bryostatin-like, phorbol ester-like, intermediate in its behavior, or more effective than either. The pattern of response, moreover, varied depending on the conditions. We conclude that the newly emerging bryostatin derivatives such as Merle 23 provide powerful tools to dissect subsets of bryostatin mechanism and response.

Keywords: Abbreviations; PKC; protein kinase C; DMSO; dimethylsulfoxide; ERK; extracellular signal-regulated kinases; MEK; MAPK/ERK kinase 1; MAPK; mitogen-activated protein kinase; JNK; cJun N-terminal kinases; PMA; phorbol 12-myristate 13-acetate; HAART; highly active antiretroviral therapy; DAG; diacylglycerol; GFP; green fluorescent protein; TNF-alpha; tumor necrosis factor alpha; MARCKS; myristoylated alanine rich C kinase substrate; TACE; TNF-alpha converting enzyme; TRAIL; TNF-related apoptosis-inducing ligandProtein kinase C; Phorbol ester; Cell signaling; Bryostatin analog

Antiproliferative and proapoptotic activity of CLM3, a novel multiple tyrosine kinase inhibitor, alone and in combination with SN-38 on endothelial and cancer cells by Guido Bocci; Anna Fioravanti; Concettina La Motta; Paola Orlandi; Bastianina Canu; Teresa Di Desidero; Laura Mugnaini; Stefania Sartini; Sandro Cosconati; Rita Frati; Alessandro Antonelli; Piero Berti; Paolo Miccoli; Federico Da Settimo; Romano Danesi (pp. 1309-1316).

To demonstrate the antiproliferative and pro-apoptotic activity of the novel pyrazolopyrimidine derivative multiple tyrosine kinase inhibitor CLM3, alone and in combination with SN-38 (the active metabolite of irinotecan), on endothelial and tumor cells and to show its mechanism of action.Proliferation and apoptotic assays were performed on microvascular endothelial (HMVEC-d) and lung (A549) and thyroid cancer (8305C, TT) cell lines exposed to CLM3 and to the simultaneous combination with SN38 for 72h. Cell-based phospho-VEGFR-2, phospho-EGFR and phospho-RET inhibition assays were performed and ERK1/2 and Akt phosphorylation were quantified by ELISA kits. Cyclin D1 gene expression was performed with real-time PCR and cyclin D1 intracellular concentrations were measured by ELISA.A strong effect on antiproliferative and pro-apoptotic activity was found with the CLM3 on endothelial and cancer cells, synergistically enhanced by SN38. Phospho-VEGFR-2, phospho-EGFR and phospho-RET levels significantly decreased after CLM3 treatments in activated endothelial and cancer cells; ERK1/2 and Akt phosphorylation were significantly inhibited by lower concentrations of the pyrazolopyrimidine drug in endothelial cells if compared to cancer cells. Moreover, CLM3 treatment greatly inhibited the expression of the cyclin D1 gene in endothelial and cancer cells, decreasing the cyclin D1 protein intracellular concentration.The pyrazolopyrimidine derivative CLM3 demonstrated a highly significant and promising antiproliferative and proapoptotic activity, alone and in combination with SN-38, for activated endothelial and cancer cells. These effects are mainly due to its inhibition of phosphorylation of VEGFR-2, EGFR and RET tyrosine kinases and their related signaling pathways.

Keywords: CLM3; Irinotecan; Angiogenesis; Synergism; Tyrosine kinase inhibitor

Sulindac sulfide induces autophagic death in gastric epithelial cells via Survivin down-regulation: A mechanism of NSAIDs-induced gastric injury by Shiun-Kwei Chiou; Neil Hoa; Amy Hodges (pp. 1317-1323).

Sulindac sulfide, a nonsteroidal anti-inflammatory drug (NSAID), has anti-tumorigenic and anti-inflammatory activities, but causes gastric mucosal damage. NSAIDs cause gastric injury in part by down-regulation of Survivin, an apoptosis inhibitor, resulting in apoptosis induction. Autophagy is a process that promotes cellular health by destroying unwanted cellular materials. Excessive autophagy induction could lead to a non-apoptotic cell death (autophagic cell death). The present study showed that sulindac sulfide at a physiological concentration also induces autophagic death in human gastric epithelial AGS and rat gastric epithelial RGM-1 cells, and that Survivin down-regulation is a mechanism involved: Sulindac sulfide treatment increased LC3b-II and APG7 levels and cytosolic vacuole formation, indications of autophagy induction, in AGS and RGM-1 cells. Sulindac sulfide treatment induced AGS and RGM-1 cell death, which was significantly reduced by pretreatment with the autophagy inhibitors 3-methyladenine and chloroquine, indicating that sulindac sulfide induced autophagic cell death. Stable overexpression of Survivin in RGM-1 cells did not inhibit the induction of LC3b-II levels or vacuole formation by sulindac sulfide, but significantly reduced the resulting cell death, suggesting that Survivin may inhibit autophagic cell death downstream of LC3b-II induction and vacuole formation. Indeed, siRNA depletion of LC3b in AGS cells inhibited the down-regulation of Survivin levels and the induction of cell death by sulindac sulfide, confirming that down-regulation of Survivin occurs in the autophagy pathway downstream of LC3b-II induction by sulindac sulfide. Induction of Survivin-dependent autophagic cell death is a novel mechanism by which sulindac sulfide induces gastric mucosal injury.

Keywords: NSAIDs; Survivin; Autophagy; Cell death; Gastric mucosal injury; Sulindac sulfide

Plant lectins are novel Toll-like receptor agonists by John Unitt; David Hornigold (pp. 1324-1328).

The T cell mitogen and plant glycoprotein, phytohaemagglutinin (PHA), is commonly used to stimulate peripheral blood mononuclear cell (PBMC) preparations to produce IL-2, IL-5, GM-CSF and IFN-γ and so provide an assay to detect immunosuppressants like FK506 and anti-inflammatories such as PDE IV inhibitors.During the early discovery of novel TLR agonists for the treatment of asthma we initially showed that PHA-L is a specific human TLR4 agonist, devoid of effects on equivalent TLR4 null cells. This TLR4 agonism was not due to LPS contamination of PHA-L, as polymyxin B was ineffective and unlike PHA-L, LPS did not stimulate TLR5 or TLR2/6. Also this specific PHA-L agonism of TLR4 was shown for different PHA forms, for example, PHA-P.This TLR lectin pharmacology finding was further explored by testing a broader panel of plant lectin representatives for agonism against a suite of hrTLR cell reporter assays (2/6, 3, 4, 5, 7, 8 and 9).Soybean agglutinin (SBA), concanavalin A (ConA) and PHA lectin family members only stimulated extracellular TLRs (2/6, 4 and 5) probably due to lack of intracellular access, whilst other lectins were either pan-active (WGA) or inactive (AIL).Interestingly SBA only stimulated TLR4, ConA, TLR2/6 and PHA-L, TLR2/6, 4 and 5. As each lectin family exhibits different sugar ligand specificity for interaction, these results suggest that the pharmacology of this TLR agonism is encoded by the lectin's carbohydrate recognition motifs and the appropriate surface presentation of these motifs on different TLRs.

Keywords: Abbreviations; LPS; lipopolysaccharide; NF-κB; nuclear factor κB; PMB; polymyxin B; ConA; concanavalin A from; Canavalia ensiformis; (jack bean); SBA; soybean agglutinin from; Glycine max; (soybean); PHA; phytohaemagglutinin from; Phaseolus vulgaris; (red kidney bean); PHA-L; tetramer of L isolectin subunits (i.e., L4); PHA-P; tetramer of L and E isolectin subunits mixtures (i.e., L1E3, L2E2, L3E, E4, L4); WGA; wheat germ agglutinin from; Triticum vulgaris; AIL; lectin from; Artocarpus integrifolia; (jacalin); PNA; peanut agglutinin from; Arachis hypogaea; IA; intrinsic activity; pEC50; −log; ₁₀; {EC; ₅₀; [LPS or lectin] (g/ml)} or −log; ₁₀; {EC; ₅₀; [compound] (M)}; NA; not activeToll-like receptors; Plant lectins; LPS; Phytohaemagglutinin; Reporter assay

Reversible epigenetic fingerprint-mediated glutathione- S-transferase P1 gene silencing in human leukemia cell lines by Tommy Karius; Michael Schnekenburger; Jenny Ghelfi; Jörn Walter; Mario Dicato; Marc Diederich (pp. 1329-1342).

Glutathione- S-transferase P1 (GSTP1) gene is commonly silenced by CpG island promoter hypermethylation in prostate, breast, and liver cancers. However, mechanisms leading to GSTP1 repression by promoter hypermethylation in leukemia and its relationship with pathological alterations of the chromatin structure remain poorly understood. A panel of leukemia cell lines was analyzed for their GSTP1 expression, revealing cell lines with high, moderate or no detectable GSTP1 expression. Bisulfite sequencing, methylation-specific PCR and combined bisulfite restriction analysis revealed that GSTP1 promoter was completely methylated in transcriptionally inactive RAJI and MEG-01 cell lines. In contrast, cell lines expressing GSTP1 exhibited an unmethylated and transcriptionally active promoter. Furthermore, histone marks and effector proteins associated with transcriptional activity were detected by chromatin immunoprecipitation in the GSTP1 expressing hypomethylated K-562 cell line. However, repressive chromatin marks and the recruitment of silencing protein complexes were found in the non-expressing hypermethylated RAJI and MEG-01 cell lines. Finally, we provide evidence that treatment of RAJI and MEG-01 cells with the DNA demethylating agent, 5-aza-2′-deoxycytidine, resulted in GSTP1 promoter demethylation, drastic changes of histone modifications and promoter associated proteins and GSTP1 gene activation. In contrast, treatments with HDAC inhibitors failed to demethylate and reactivate the GSTP1 gene. Our study extends the knowledge on leukemia-specific epigenetic alterations of GSTP1 gene. Furthermore, we are showing the correlation of DNA methylation and histone modifications with the positive/negative GSTP1 transcriptional expression state. Finally, these data support the concept of the dominance of DNA methylation over HDAC inhibitor-sensitive histone deacetylation in gene silencing.

Keywords: GSTP1; Leukemia; DNA methylation; Chromatin; Epigenetic silencing

Resveratrol exerts anti-obesity effects via mechanisms involving down-regulation of adipogenic and inflammatory processes in mice by Soyoung Kim; Yoojeong Jin; Youngshim Choi; Taesun Park (pp. 1343-1351).

Resveratrol is a natural polyphenolic stilbene derivative found in a variety of edible fruits, including nuts, berries, and grape skin. Although resveratrol has been suggested to improve thermogenesis in the brown adipose tissues of obese animals, there have been no reports on the anti-adipogenic and anti-inflammatory effects of resveratrol in the white adipose tissues of obese animals. The primary aim of this study was to investigate whether resveratrol attenuates high-fat diet (HFD)-induced adipogenesis and inflammation in the epididymal fat tissues of mice and to explore the underlying mechanisms involved in this attenuation. In comparison with HFD-fed mice, mice fed with a 0.4% resveratrol-supplemented diet (RSD) showed significantly lower body weight gain (−48%), visceral fat-pad weights (−58%), and plasma levels of triglyceride, FFA, total cholesterol, glucose, tumor necrosis factor (TNF) α, and monocyte chemoattractant protein-1 (MCP1). Resveratrol significantly reversed the HFD-induced up-regulation of galanin-mediated signaling molecules (GalR1/2, PKCδ, Cyc-D, E2F1, and p-ERK) and key adipogenic genes (PPARγ2, C/EBPα, SREBP-1c, FAS, LPL, aP2, and leptin) in the epididymal adipose tissues of mice. Furthermore, resveratrol significantly attenuated the HFD-induced up-regulation of pro-inflammatory cytokines (TNFα, IFNα, IFNβ, and IL-6) and their upstream signaling molecules (TLR2/4, MyD88, Tirap, TRIF, TRAF6, IRF5, p-IRF3, and NF-κB) in the adipose tissues of mice. The results of this study suggest that resveratrol inhibits visceral adipogenesis by suppressing the galanin-mediated adipogenesis signaling cascade. It may also attenuate cytokine production in the adipose tissue by repressing the TLR2- and TLR4-mediated pro-inflammatory signaling cascades in HFD-fed mice.

Keywords: Abbreviations; aP2; adipocyte protein 2; C/EBPα; CCAAT/enhancer binding protein alpha; RSD; resveratrol-supplemented diet; Cyc-D; cyclin D; DIO; diet induced obesity; E2F1; E2F transcription factor 1; FAS; fatty acid synthase; FFAs; free fatty acids; GalR1; galanin receptor 1; GalR2; galanin receptor 2; GAPDH; glyceraldehydes 3-phosphate dehydrogenase; HFD; high-fat diet; IFNα; interferon-alpha; IFNβ; interferon-beta; IL-6; interleukin 6; IRF5; interferon regulatory factor 5; LXR; liver X receptor; LPL; lipoprotein lipase; MCP1; monocyte chemoattractant protein-1; MyD88; myeloid differentiation primary response gene 88; ND; normal diet; NF-κB; nuclear factor-kappaB; PKCδ; protein kinase C delta; PPARγ2; peroxisome proliferator-activated receptor gamma 2; SREBP-1c; sterol regulatory element-binding factor 1; Tirap; toll-interleukin 1 receptor (TIR) domain-containing adaptor protein; TLR2; toll like receptor 2; TLR4; toll like receptor 4; TNFα; tumor necrosis factor-alpha; TRAF6; TNF receptor-associated factor 6; TRIF; TIR-domain-containing adapter-inducing interferon-βAnti-obesity; Resveratrol; High-fat diet; Adipogenesis; Inflammation

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Biochemical Pharmacology (v.81, #11)