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Biochemical Pharmacology (v.81, #10)
Exosomes as intercellular signalosomes and pharmacological effectors by Michel Record; Caroline Subra; Sandrine Silvente-Poirot; Marc Poirot (pp. 1171-1182).
Cell secretion is a general process involved in various biological responses. Exosomes are part of this process and have gained considerable scientific interest in the past five years. Several steps through investigations across the last 20years can explain this interest. First characterized during reticulocyte maturation, they were next evidenced as a key player in the immune response and cancer immunotherapy. More recently they were reported as vectors of mRNAs, miRNAs and also lipid mediators able to act on target cells. They are the only type of vesicles released from an intracellular compartment from cells in viable conditions. They appear as a vectorized signaling system operating from inside a donor cell towards either the periphery, the cytosol, or possibly to the nucleus of target cells. Exosomes from normal cells trigger positive effects, whereas those from pathological ones, such as tumor cells or infected ones may trigger non-positive health effects. Therefore regulating the biogenesis and secretion of exosomes appear as a pharmacological challenge to intervene in various pathophysiologies. Exosome biogenesis and molecular content, interaction with target cells, utilisation as biomarkers, and functional effects in various pathophysiologies are considered in this review.
Keywords: Cancer; HIV; LBPA or Bis(monoacylglycero)phosphate; Prostaglandine
The angiogenic process as a therapeutic target in cancer by Esther M. Bridges; Adrian L. Harris (pp. 1183-1191).
Angiogenesis has emerged as a critical process for tumour progression. Identifying key pathways involved in the regulation and promotion of angiogenesis has resulted in the development of numerous approaches targeting pro-angiogenic signalling pathways. The most prominent and characterised pro-angiogenic pathway is the vascular endothelial growth factor signalling pathway. This review will describe several inhibitors of angiogenesis currently in clinical trial and their various targets. Targeting pro-angiogenic pathways has improved outcome for many patients, however, the emerging problems with drug resistance with clinically approved angiogenic inhibitors will also be discussed in this review. It is hoped that identifying the causes of tumour re-growth and disease progression following treatment will enable future anti-angiogenic therapies to circumvent resistance.
Keywords: Abbreviations; VEGF; vascular endothelial growth factor; VEGFR; vascular endothelial growth factor receptor; FGF2; fibroblast growth factor 2; HGF; hepatocyte growth factor; PDGF; platelet derived endothelial growth factor; PIGF; placental growth factor; Dll4; delta like-4Anti-angiogenic therapy; Bevacizumab; Biomarkers; Resistance
Cudratricusxanthone G inhibits human colorectal carcinoma cell invasion by MMP-2 down-regulation through suppressing activator protein-1 activity by Lisha Kuang; Lei Wang; Qian Wang; Qufei Zhao; Bing Du; Dali Li; Jian Luo; Mingyao Liu; Aijun Hou; Min Qian (pp. 1192-1200).
Cudratricusxanthone G (CTXG), a natural bioactive cudratricusxanthone extracted from C. tricuspidata, has shown anti-cancer properties. However, the function and mechanism of CTXG in tumor invasion have not been elucidated to date. In this study, we investigated the inhibitory effect of CTXG on the proliferation, migration and invasion of SW620 cells. We found that MMP-2, a pivotal factor in tumor invasion, was suppressed in both expression and activation by CTXG in a dose-dependent manner. The suppression of MMP-2 expression by CTXG led to an inhibition of SW620 cells invasion and migration by inactivating Rac1 and Cdc42 but not RhoA GTPase. Furthermore, CTXG also inhibited the transcriptional activity of AP-1 (activator protein-1). In conclusion, our data demonstrate that CTXG exerted anti-invasion action in SW620 cells by targeting MMP-2 though regulating the activities of Rac1, Cdc42 and their downstream transcriptional factor AP-1. These results are the first to reveal the novel functions of CTXG in cancer cell invasion and its molecular basis for the anti-cancer action.
Keywords: AP-1; Cudratricusxanthone G (CTXG); Invasion; MMP-2; Rac1
KS900: A hypoxia-directed, reductively activated methylating antitumor prodrug that selectively ablates O6-alkylguanine-DNA alkyltransferase in neoplastic cells by Raymond P. Baumann; Kimiko Ishiguro; Philip G. Penketh; Krishnamurthy Shyam; Rui Zhu; Alan C. Sartorelli (pp. 1201-1210).
To most effectively treat cancer it may be necessary to preferentially destroy tumor tissue while sparing normal tissues. One strategy to accomplish this is to selectively cripple the involved tumor resistance mechanisms, thereby allowing the affected anticancer drugs to gain therapeutic efficacy. Such an approach is exemplified by our design and synthesis of the intracellular hypoxic cell activated methylating agent, 1,2-bis(methylsulfonyl)-1-methyl-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS900) that targets the O-6 position of guanine in DNA. KS900 is markedly more cytotoxic in clonogenic experiments under conditions of oxygen deficiency than the non-intracellularly activated agents KS90, and 90M, when tested in O6-alkylguanine-DNA alkyltransferase (AGT) non-expressing cells (EMT6 mouse mammary carcinoma, CHO/AA8 hamster ovary, and U251 human glioma), and than temozolomide when tested in AGT expressing cells (DU145 human prostate carcinoma). Furthermore, KS900 more efficiently ablates AGT in HL-60 human leukemia and DU145 cells than the spontaneous globally activated methylating agent KS90, with an IC50 value over 9-fold lower than KS90. Finally, KS900 under oxygen-deficient conditions selectively sensitizes DU145 cells to the chloroethylating agent, onrigin, through the ablation of the resistance protein AGT. Thus, under hypoxia, KS900 is more cytotoxic at substantially lower concentrations than methylating agents such as temozolomide that are not preferentially activated in neoplastic cells by intracellular reductase catalysts. The necessity for intracellular activation of KS900 permits substantially greater cytotoxic activity against cells containing the resistance protein O6-alkylguanine-DNA alkyltransferase (AGT) than agents such as temozolomide. Furthermore, the hypoxia-directed intracellular activation of KS900 allows it to preferentially ablate AGT pools under the oxygen-deficient conditions that are present in malignant tissue.
Keywords: Abbreviations; AGT; O; 6; -alkylguanine-DNA alkyltransferase; 90CE; 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine; 90M; 1,2-bis(methylsulfonyl)-1-methyl-2-(methylamino)carbonylhydrazine; DCM; dichloromethane; KS900; 1,2-bis(methylsulfonyl)-1-methyl-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine; KS119; 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine; Onrigin™; 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylamino)carbonylhydrazine; O; 6; -BG; O; 6; -benzylguanineOxygen-deficient cells; O; 6; -alkylguanine-DNA alkyltransferase; 1,2-Bis(sulfonyl)hydrazines; KS900; Onrigin™
4-Methylcatechol-induced oxidative stress induces intrinsic apoptotic pathway in metastatic melanoma cells by Florastina Payton; Rumu Bose; William L. Alworth; Addanki P. Kumar; Rita Ghosh (pp. 1211-1218).
There has been a steady rise in fatalities associated with thick melanomas (>4mm). Although understanding of the biology of the disease has improved, effective treatment strategies for patients with advanced metastatic melanoma remain elusive. Therefore, more intensive testing of agents with therapeutic potential are needed to improve survival of patients with metastatic malignant melanoma. We have tested the ability of 4-methylcatechol, a metabolite of quercetin; a naturally occurring compound that is commonly found in a variety of fruits for its potential as an anti-melanoma agent. Our results show that 4-methylcatechol inhibits proliferation of melanoma cells in culture while not affecting the growth of normal human epidermal melanocytes. Further, the ability of metastatic melanoma cells to form colonies on soft agar was also inhibited. 4-Methylcatechol caused the accumulation of cells in G2/M phase of the cell cycle and induced apoptosis. There was an increase in reactive oxygen species following treatment with 4-methylcatechol that led to apoptosis through the intrinsic mitochondrial pathway. Treatment also inhibited cell survival mediated by Akt, a key player in melanoma cell survival. Taken together our results suggest that 4-methylcatechol exhibits cytotoxicity towards metastatic malignant melanoma cells while sparing normal melanocytes and should be tested further as a potential drug candidate for malignant melanoma.
Keywords: Melanoma; 4-Methylcatechol; Reactive oxygen species; Cell cycle arrest; Apoptosis; Survival
Extracellular nucleotide derivatives protect cardiomyocytes against hypoxic stress by O. Golan; Y. Issan; A. Isak; J. Leipziger; B. Robaye; A. Shainberg (pp. 1219-1227).
Hypoxia causes cardiac cell damage. Extracellular nucleotides derivatives or di/triphosphate protects the cardiomyocytes from hypoxic damage.Extracellular nucleotides have widespread effects and various cell responses. Whereas the effect of a purine nucleotide (ATP) and a pyrimidine nucleotide (UTP) on myocardial infarction has been examined, the role of different purine and pyrimidine nucleotides and nucleosides in cardioprotection against hypoxic stress has not been reported.To investigate the role of purine and pyrimidine nucleotides and nucleosides in protective effects in cardiomyocytes subjected to hypoxia.Rat cultured cardiomyocytes were treated with various extracellular nucleotides and nucleosides, before or during hypoxic stress. The results revealed that GTP or CTP exhibit cardioprotective ability, as revealed by lactate dehydrogenase (LDH) release, by propidium iodide (PI) staining, by cell morphology, and by preserved mitochondrial activity. Pretreatment with various P2 antagonists (suramin, RB-2, or PPADS) did not abolish the cardioprotective effect of the nucleotides. Moreover, P2Y2−/−, P2Y4−/−, and P2Y2−/−/P2Y4−/− receptor knockouts mouse cardiomyocytes were significantly protected against hypoxic stress when treated with UTP. These results indicate that the protective effect is not mediated via those receptors. We found that a wide variety of triphosphate and diphosphate nucleotides (TTP, ITP, deoxyGTP, and GDP), provided significant cardioprotective effect. GMP, guanosine, and ribose phosphate provided no cardioprotective effect. Moreover, we observed that tri/di-phosphate alone assures cardioprotection. Treatment with extracellular nucleotides, or with tri/di-phosphate, administered under normoxic conditions or during hypoxic conditions, led to a decrease in reactive oxygen species production.Extracellular tri/di-phosphates are apparently the molecule responsible for cardioprotection against hypoxic damage, probably by preventing free radicals formation.
Keywords: Abbreviations; DCFH-DA; 2′,7′-dichlorofluorescein-diacetate; 4-Di-1-ASP; =; DASPMI; 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide; E-NPP; ecto-nucleotide pyro-phosphatase; LDH; lactate dehydrogenase; NDPs; nucleotide diphosphates; NMP; nucleotide monophosphate; NTPs; nucleotide triphosphates; RB-2; Reactive blue 2; PBS; phosphate buffer saline; PI; propidium iodide; PPADS; pyridoxal-phosphate-6-azophenyl-2,4-disulfonate; PPi; inorganic pyro-phosphate; RDB; a solution of proteolytic enzymes; ROS; reactive oxygen species; SDH; Succinate dehydrogenase; WT; wild typeCardioprotection; Pyrimidines; Purines; Pyrophosphate; ROS
MG132 treatment attenuates cardiac remodeling and dysfunction following aortic banding in rats via the NF-κB/TGFβ1 pathway by Yuedong Ma; Baolin Chen; Dan Liu; Yang Yang; Zhaojun Xiong; Junyi Zeng; Yugang Dong (pp. 1228-1236).
Although MG132, a proteasome inhibitor, is suggested to impede secondary cardiac remodeling after hypertension, the mechanism and optimal duration of treatment remain unknown. This study was designed to investigate the effects and possible mechanism of MG132 on hypertension-induced cardiac remodeling. Male Sprague–Dawley rats subjected to abdominal aortic constriction (AAC) or sham operation received an intraperitoneal injection of MG132 (0.1mgkg−1day−1) or vehicle over a 2- or 8-week period. In the end, left ventricular (LV) function was evaluated with echocardiography and pressure tracing. Collagen deposition within the LV myocardium was assessed with Masson's trichrome staining. Ubiquitin-proteasome system (UPS), NF-κB, I-κB, TGFβ1 and Smad2 within the LV tissue were evaluated. In addition, angiotensin II within both plasma and LV tissue was also examined. Compared with the sham groups, the vehicle-treated AAC group exhibited a higher angiotensin II level, LV/body weight ratio, septal and posterior wall thicknesses, and a markedly reduced cardiac function ( P<0.05). Treatment with MG132 for 8 weeks attenuated these cardiac remodeling parameters and improved cardiac function ( P<0.01). 2- and 8-week hypertension led to activation of UPS, which was followed by activation of NF-κB and increased expression of TGFβ1 and Smad2 ( P<0.01). MG132 significantly inhibited NF-κB activity and down-regulate the levels of TGFβ1 and Smad2 expression by 2 and still at 8 weeks ( P<0.01). Short- and long-term treatment with MG132 significantly attenuated hypertension-induced cardiac remodeling and dysfunction, which may be mediated by the NF-κB/TGFβ1 signaling pathway.
Keywords: Abbreviations; AAC; abdominal aortic constriction; TAC; transverse aortic constriction; LV; left ventricle; UPS; ubiquitin-proteasome system; CHF; congestive heart failure; Ang II; angiotensin II; MAPK; mitogen-activated protein kinase; BW; body weight; LVW; left ventricular weight; LV/BW; left ventricular/body weight ratio; Lung/BW; lung/body weight ratio; HR; heart rate; LVSP; left ventricular systolic pressure; LVEDP; left ventricular end-diastolic pressure; +dP/dtmax; the maximal rate of LV contraction; −dP/dtmax; the maximal rate of LV relaxation; IVSd; interventricular septal thickness; PWd; left ventricular posterior wall thickness; LVDd; left ventricular end-diastolic diameter; FS; fractional shortening; EF; ejection fractionPressure overload; Proteasome inhibition; Cardiac remodeling; Heart failure; NF-κB
A farnesylated G-protein suppresses Akt phosphorylation in INS 832/13 cells and normal rat islets: Regulation by pertussis toxin and PGE2 by Chandrashekara N. Kyathanahalli; Anjaneyulu Kowluru (pp. 1237-1247).
Proposed model for Probin-mediated suppression of PKB/Akt signaling pathway in pancreatic β-cells.Protein isoprenylation constitutes incorporation of either 15-carbon farnesyl or 20-carbon geranylgeranyl derivative of mevalonic acid onto the C-terminal cysteine, culminating in increased hydrophobicity of the modified proteins for optimal membrane anchoring and interaction with their respective effectors. Emerging evidence confirms the participatory role of prenylated proteins in pancreatic β-cell function including insulin secretion. Herein, we investigated the putative regulatory roles of protein farnesylation in cell survival signaling pathways in insulin-secreting INS 832/13 cells and normal rodent islets, specifically at the level of protein kinase-B/Akt phosphorylation induced by insulin-like growth factor [IGF-1]. Selective inhibitors of farnesylation [e.g., FTI-277 or FTI-2628] or knockdown of the β-subunit of farnesyl transferase by siRNA significantly increased Akt activation under basal and IGF-1-stimulated conditions. Consequentially, the relative abundance of phosphorylated FoxO1 and Bad were increased implicating inactivation of critical components of the cell death machinery. In addition, FTI-induced Akt activation was attenuated by the PI3-kinase inhibitor, LY294002. Exposure of INS 832/13 cells to pertussis toxin [PTx] markedly potentiated Akt phosphorylation suggesting involvement of a PTx-sensitive G-protein in this signaling axis. Furthermore, prostaglandin E2, a known agonist of inhibitory G-proteins, significantly attenuated FTI-induced Akt phosphorylation. Taken together, our findings suggest expression of a farnesylated G-protein in INS 832/13 cells and normal rat islets, which appear to suppress Akt activation and subsequent cell survival signaling steps. Potential regulatory roles of the islet endogenous protein kinase-B inhibitory protein [Probin] in islet function are discussed.
Keywords: Abbreviations; Erk; extracellular signal regulated kinase; FoxO1; Forkhead box protein O1; FTI; farnesyl transferase inhibitor; FTase; farnesyl transferase; GTP; guanosine triphosphate; IGF-1; insulin-like growth factor-1; MEK; mitogen activated extracellular kinase; MPA; mycophenolic acid; PI3-kinase; phosphoinositide 3 phosphate kinase; PKB; protein kinase B; PGE; 2; prostaglandin E; 2; PTx; pertussis toxin; siRNA; small interfering RNAPancreatic β-cells; Protein farnesylation; Akt; FoxO, β-Cell survival
Distinct pharmacological properties of morphine metabolites at Gi-protein and β-arrestin signaling pathways activated by the human μ-opioid receptor by Nadine Frölich; Christian Dees; Christian Paetz; Xuan Ren; Martin J. Lohse; Viacheslav O. Nikolaev; Meinhart H. Zenk (pp. 1248-1254).
Morphine and several other opioids are important drugs for the treatment of acute and chronic pain. Opioid-induced analgesia is predominantly mediated by the μ-opioid receptor (MOR). When administered to humans, complex metabolic pathways lead to generation of many metabolites, nine of which may be considered major metabolites. While the properties of the two main compounds, morphine-6-glucuronide and morphine-3-glucuronide, are well described, the activity of other morphine metabolites is largely unknown. Here we performed an extensive pharmacological characterization by comparing efficacies and potencies of morphine and its nine major metabolites for the two main signaling pathways engaged by the human MOR, which occur via Gi-protein activation and β-arrestins, respectively. We used radioligand binding studies and FRET-based methods to monitor MOR-mediated Gi-protein activation and β-arrestin recruitment in single intact 293T cells. This approach identified two major groups of morphine metabolites, which we classified into “strong” and “weak” receptor ligands. Strong partial agonists morphine, morphine-6-glucuronide, normorphine, morphine-6-sulfate, 6-acetylmorphine and 3-acetylmorphine showed efficacies in the nanomolar range, while the weak metabolites morphine-N-oxide, morphine-3-sulfate, morphine-3-glucuronide and pseudomorphine activated MOR pathways only in the micromolar range. Interestingly, three metabolites, normorphine, 6-acetylmorphine and morphine-6-glucuronide, had lower potencies for Gi-protein activation but higher potencies and efficacies for β-arrestin recruitment than morphine itself, suggesting that they are biased towards β-arrestin pathways.
Keywords: Abbreviations; DAMGO; [; d; -Ala; 2; , N-MePhe; 4; , Gly-ol]-enkephalin; FRET; fluorescence (or: Förster) resonance energy transfer; GPCR; G-protein-coupled receptor; GRK; G-protein-coupled-receptor kinaseMorphine; Metabolite; FRET; G-protein; β-Arrestin
Critical selection of reliable reference genes for gene expression study in the HepaRG cell line by Liesbeth Ceelen; Ward De Spiegelaere; Michael David; Jurgen De Craene; Mathieu Vinken; Tamara Vanhaecke; Vera Rogiers (pp. 1255-1261).
The human HepaRG cell line has shown to be a valuable in vitro tool for repeated exposure to chemical compounds and to evaluate their potential toxic outcome. Seen the importance given by the actual EU legislation of cosmetics and chemical substances to the use of in vitro methods in human safety evaluation, one can expect that HepaRG cells will gain importance as human-relevant cell source. At the transcriptional level, RT-qPCR assays are often used to obtain quantitative results. The choice of internal control is important since it may affect the study outcome. Indeed, it is well-known that expression levels of traditional reference genes can vary across tissue types and across experimental settings within one specific tissue type. From a review of the scientific literature, it appears that, for HepaRG cells, S18 often is used as internal control, but without any evidence of its expression stability in this cell line. Therefore, we aimed to select the most optimal reference genes for gene expression studies in HepaRG cells and to check whether S18 is a suitable reference gene. Twelve candidate genes’ expression stability level was analyzed by three algorithms (geNorm, BestKeeper, Normfinder), which identified the optimal single reference gene ( TBP) and the most suitable set of reference genes ( TBP, UBC, SDHA, RLP13, YHWAZ, HMBS, B2M and HPRT1) for HepaRG transcriptional profiling. This study provides a new set of reference genes that is suitable for testing whenever RT-qPCR data for HepaRG cells are generated. The most stable ones can then be selected for further normalization.
Keywords: HepaRG; RT-qPCR; Reference gene; Normalization