Check out our New Publishers' Select for Free Articles

Biochemical Pharmacology (v.73, #7)

Editorial Advisory Board (pp. iii).

Acyclic nucleoside phosphonates: Past, present and future by E. De Clercq (pp. 911-922).

Twenty years following the description of the broad-spectrum antiviral activity of S-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [( S)-HPMPA] [De Clercq E, Holý A, Rosenberg I, Sakuma T, Balzarini J, Maudgal PC. A novel selective broad-spectrum anti-DNA virus agent. Nature 1986;323:464–7], the acyclic nucleoside phosphonates have acquired a prominent therapeutic position: (i) cidofovir in the treatment of papilloma-, herpes-, adeno- and poxvirus infections, (ii) adefovir in the treatment of chronic hepatitis B virus (HBV) infections, and (iii) tenofovir in the treatment of human immunodeficiency virus (HIV) infections (AIDS). Although formally approved only for the treatment of human cytomegalovirus (HCMV) retinitis in AIDS patients, cidofovir has been used successfully in the treatment of various other DNA virus infections, particularly human papilloma virus (HPV)-associated lesions. Adefovir dipivoxil has become a standard therapy for HBV infections, especially when resistant to lamivudine. Tenofovir disoproxil fumarate (TDF) is the corner stone of the triple-drug (TDF, emtricitabine, and efavirenz) combination therapy for AIDS, and TDF, alone or combined with emtricitabine may in the future evolve to the standard therapy of hepatitis B. Guided by the results obtained with tenofovir in the prevention of parenteral, intravaginal and perinatal infections with simian immunodeficiency virus in monkeys, and the safety profile gathered with TDF in humans with AIDS over the past 5 years since TDF was licensed for clinical use, it should be further pursued for the pre- and post-exposure prophylaxis of HIV infections in humans. Meanwhile, new classes of both acyclic (i.e. PMPO-DAPy, PMEO-DAPy, HPMPO-DAPy) and cyclic nucleoside phosphonates (i.e. PMDTA, PMDTT, GS9148) have been accredited with an antiviral potency and selectivity similar to those of cidofovir, adefovir and/or tenofovir.

Keywords: Acyclic nucleoside phosphonates; Nucleotide analogues; Cidofovir; Adefovir; Tenofovir; Antiviral activity

Induction of CYP2C12 expression in senescent male rats is well correlated to an increase of HNF3β expression, while the decline of CYP2C11 expression is unlikely due to a decrease of STAT5 activation by Valérie Wauthier; Pascaline Dubois; Roger K. Verbeeck; Pedro Buc Calderon (pp. 923-933).

Ageing affects drugs metabolism influencing the therapeutic efficacy and safety of drugs. By using the experimental model of aged male rats, we investigated the influence of ageing on some CYP2C isoforms, the most important CYP450 sub-family in rats. The activity of the male specific CYP2C11 is decreased by 55% in senescent male rats. This correlates with a significant reduction of both protein content (80%) and mRNA (60%) indicating a demasculinization process. The expression of CYP2C12, a female specific isoform, is induced in senescent male rats indicating a feminization process. Neither the activity nor the expression of CYP2C6, a female predominant isoform, is modified in senescent male rats. Thereafter, certain putative GH mediators like some liver enriched transcription factors (LETFs) or STAT5b were investigated. The amount of HNF3β mRNA, a transcription factor involved in the up-regulation of CYP2C12, has been shown to increase by about three-fold in senescent male rats. With regard to STAT5b, which has been reported to be involved in the male specific regulation of CYP2C11, large amounts of phosphorylated STAT5 were observed in the liver of senescent male rats. These results indicate that while the induction of CYP2C12 during ageing could be due, at least partially, to the enhanced HNF3β expression, the decline of CYP2C11 is unlikely related to a decrease of STAT5 activation.

Keywords: Abbreviations; BSA; bovine serum albumin; CYP; cytochrome P450; GH; growth hormone; LETF; liver enriched transcription factor; HNF; hepatocyte nuclear factor; JAK; Janus tyrosine kinase; STAT; signal transducer and activator of transcriptionCYP2C11; CYP2C12; Ageing; Growth hormone; LETFs and STAT5b

Circumvention of the multidrug-resistance protein (MRP-1) by an antitumor drug through specific inhibition of gene transcription in breast tumor cells by Sylvia Mansilla; Marta Rojas; Marc Bataller; Waldemar Priebe; José Portugal (pp. 934-942).

Multidrug-resistance protein 1 (MRP-1) confers resistance to a number of clinically important chemotherapeutic agents. The promoter of the mrp-1 gene contains an Sp1-binding site, which we targeted using the antitumor bis-anthracycline WP631. When MCF-7/VP breast cancer cells, which overexpress MRP-1 protein, were incubated with WP631 the expression of the multidrug-resistance protein gene decreased. Conversely, doxorubicin did not alter mrp-1 gene expression. The inhibition of gene expression was followed by a decrease in the activity of the MRP-1 protein. The IC₇₅ for WP631 (drug concentration required to inhibit cell growth by 75%) circumvented the drug-efflux pump, without addition of resistant modifiers. After treatment with WP631, MCF-7/VP cells were committed to die after entering mitosis (mitotic catastrophe), while treatment with doxorubicin did not affect cell growth. This is the first report on an antitumor drug molecule inhibiting the mrp-1 gene directly, rather than being simply a poor substrate for the transporter-mediated efflux. However, both situations appeared to coexist, thereby a superior cytotoxic effect was attained. Ours results suggest that WP631 offers great potential for the clinical treatment of tumors displaying a multidrug-resistance phenotype.

Keywords: Abbreviations; CFSE; carboxyfluorescein diacetate succinimidyl ester; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; MDR; multidrug resistance; MRP-1; multidrug-resistance associate protein-1; PCR; polymerase chain reaction; qRT-PCR; quantitative real-time PCRMultidrug resistance; MRP-1; Breast cancer; WP631; Doxorubicin; Polyploidy

G1 cell cycle arrest by amlodipine, a dihydropyridine Ca²⁺ channel blocker, in human epidermoid carcinoma A431 cells by Junko Yoshida; Takaharu Ishibashi; Matomo Nishio (pp. 943-953).

We demonstrated previously that amlodipine, a dihydropyridine Ca²⁺ channel blocker, exhibits antitumor effects on human epidermoid carcinoma A431 cells both in vitro and in vivo, in part through inhibition of capacitative Ca²⁺ entry. In this study, we examined the effects of amlodipine on cell cycle distribution and cell cycle regulatory molecules in A431 cells, since a rise in intracellular Ca²⁺ is required at several points during cell cycle progression. Flow cytometric analysis revealed that treatment with amlodipine (20–30μM, for 24h) induced G1 phase cell accumulation. The amlodipine-induced G1 arrest was associated with a decrease in phosphorylation of retinoblastoma protein (pRB), a regulator of G1 to S phase transition, reduction of protein levels of cyclin D1 and cyclin dependent kinase 4 (CDK4), G1 specific cell cycle proteins, and increased expression of p21^Waf1/Cip1, an inhibitory protein of CDK/cyclin complexes. In vitro kinase assay revealed that amlodipine significantly decreased CDK2-, CDK4-, and their partners cyclin E- and cyclin D1-associated kinase activities. The amlodipine-induced reductions in cyclin D1 protein expression and in CDK2 kinase activity were reproduced by a dihydropyridine derivative, nicardipine, having an inhibitory effect on A431 cell growth, but not by nifedipine, lacking the antiproliferative activity. Our results demonstrate that amlodipine caused G1 cell cycle arrest and growth inhibition in A431 cells through induction of p21^Waf1/Cip1 expression, inhibition of CDK/cyclin-associated kinase activities, and reduced phosphorylation of pRB.

Keywords: Calcium channel blocker; G1 cell cycle arrest; pRB; p21; ^Waf1/Cip1; Cyclin dependent kinase; Human epidermoid carcinoma A431 cells

Biochemical and pharmacological characterization of P-site inhibitors on homodimeric guanylyl cyclase domain from natriuretic peptide receptor-A by Simon Joubert; Normand McNicoll; André De Léan (pp. 954-963).

Guanylyl cyclases catalyze the formation of cGMP from GTP. This family of enzymes includes soluble (sGC) and particulate guanylyl cyclases (pGC). The sGC are heterodimers containing one active catalytic site and one inactive pseudo-site. They are activated by nitric oxide. The pGC are homodimers whose activity is notably regulated by peptide binding to the extracellular domain and by ATP binding to the intracellular kinase homology domain (KHD). The catalytic mechanism of the pGC is still not well understood. Homology modeling of the structure of the homodimeric guanylyl cyclase domain, based on the crystal structure of adenylyl cyclase, suggests the existence of two functional sites for the substrate GTP. We used a purified and fully active recombinant catalytic domain from mammalian pGC, to document its enzyme kinetics properties in the absence of the KHD. The enzyme presents positive cooperativity with the substrate Mg-GTP. However, a heterodimeric catalytic domain mutant (GC-HET) containing only one active catalytic site is non-cooperative and is more similar to sGC. Structure–activity studies of purine nucleoside analogs indicate that 2′d3′GMP is the most potent inhibitor of pGC tested. It displays mixed non-competitive inhibition properties that are potentiated by the second catalytic product inorganic pyrophosphate (PPi). It appears to be equivalent to purinergic site (P-site) inhibitors characterized on particulate adenylyl cyclase. Inhibition of pGC by 2′d3′GMP in the presence of PPi is accompanied by a loss of cooperative enzyme kinetics. These results are best explained by an allosteric dimer model with positive cooperativity for both the substrate and inhibitors.

Keywords: Abbreviations; sGC; soluble guanylyl cyclase; pGC; particulate guanylyl cyclase; PPi; inorganic pyrophosphate; 2′d3′GMP; 2′-deoxy-3′-guanosine monophosphate; 2′d5′AMP; 2′-deoxy-5′-adenosine monophosphate; 2′d5′GMP; 2′-deoxy-5′-guanosine monophosphate; 2′5′dd3′AMP; 2′5′-dideoxyadenosine-3′-monophosphate; GC-A; guanylyl cyclase A; NPR-A; natriuretic peptide receptor-A; GC-C; guanylyl cyclase C or guanylin/enterotoxin receptor; ANP; atrial natriuretic peptide; GC-E; guanylyl cyclase E; retGC-1; retinal guanylyl cyclase; KHD; kinase homology domain; k; _cat; catalytic constant; S; _0.5; Michaelis–Menten constant; K; _i; inhibition or dissociation constant of inhibitor; V; _max; maximal enzyme activityGuanylyl cyclase; Natriuretic peptide; P-site inhibitor; Nucleotide; Purine; Catalysis; Protein dimer

Conformational state of human cardiac 5-HT_4(g) receptors influences the functional effects of polyclonal anti-5-HT₄ receptor antibodies by Emmanuella Di Scala; Stéphanie Rose; Olivier Hérault; Jorge Argibay; Pierre Cosnay; Véronique Bozon (pp. 964-971).

The functional effects of the anti-G21V antibody directed against the second extracellular loop of human heart 5-HT₄ receptors can differ when the receptors are expressed in different cell lines. Here, we extend these studies to show variation in the responses of 5-HT_4(g) receptors to the antibody within the same expression system. In a previous report no effect of the anti-G21V antibodies had been shown upon 5-HT_4(g) receptors expressed in CHO cells. Here the same antibodies alone or when added before 5-HT had a functional “inverse-agonist like” effect upon 5-HT_4(g) receptors expressed in a separate line of CHO cells. Although these CHO cells showed a lower efficacy of cAMP production evoked by 5-HT than the previous report they express a similar h5-HT_4(g) receptor density. Inhibition of either phosphodiesterases or Gi proteins had no effect upon the action of the antibody. Conformational states of the 5-HT₄ receptor and/or equilibrium between different states of receptors may then determine the functional effect of antibodies against this receptor.

Keywords: Antibody; Conformational state; Inverse-agonist; 5-HT; ₄; receptor; Serotonin; CHO cells

Production of extracellular superoxide by human lymphoblast cell lines: Comparison of electron spin resonance techniques and cytochrome C reduction assay by Sergey I. Dikalov; Wei Li; Payam Mehranpour; Shaoshan S. Wang; A. Maziar Zafari (pp. 972-980).

Superoxide production by NADPH oxidases plays an important role in the development and progression of cardiovascular disease (CVD). However, measurement of superoxide (O₂⁻), a marker of oxidative stress, remains a challenging task in clinical and translational studies. In this study we analyzed O₂⁻ production in cultured human lymphoblast cell lines by three different methods: (a) superoxide dismutase (SOD)-inhibitable cytochrome C reduction, (b) spin trapping of superoxide with 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide (EMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), and (c) using electron spin resonance (ESR) with the cell-permeable spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH). Lymphocytes were isolated and immortalized by an Epstein–Barr Virus (EBV)-transformation procedure. Superoxide was measured in cultured lymphoblast cell lines at baseline and upon stimulation with phorbol 12-myristate 13-acetate (PMA). Cytochrome C and the spin traps EMPO and DEPMPO detected two to five times less superoxide compared to CMH. Thus, CMH provided the most quantitative measurement of superoxide generation in human lymphoblast cell lines. Superoxide detection with CMH was linear dependent on cell concentration and was inhibited by SOD but not by catalase. Both cell-permeable polyethylene glycol (PEG)–SOD and extracellular Cu,Zn–SOD inhibited O₂⁻ detection by 90% in PMA-stimulated cells, suggesting a predominantly extracellular O₂⁻ generation in human lymphoblasts. Our study describes a new technique for O₂⁻ measurement in cultured human lymphoblasts using ESR and CMH. A highly sensitive in vitro measurement of O₂⁻ in human cell lines would allow investigators to study genotype/phenotype interactions in translational studies.

Keywords: Cardiovascular disease; NADPH oxidase; Superoxide; Electron spin resonance; Spin trap; Spin probe; Cytochrome; C

Structure and function of eritadenine and its 3-deaza analogues: Potent inhibitors of S-adenosylhomocysteine hydrolase and hypocholesterolemic agents by Taro Yamada; Junichi Komoto; Kaiyan Lou; Akiharu Ueki; Duy H. Hua; Kimio Sugiyama; Yoshimi Takata; Hirofumi Ogawa; Fusao Takusagawa (pp. 981-989).

d-Eritadenine (DEA) is a potent inhibitor of S-adenosyl-l-homocysteine hydrolase (SAHH) and has hypocholesterolemic activity. We have hypothesized that 3-deaza-DEA (C3-DEA) and its analogues retain high level of SAHH inhibitory activity and have resistance to deamination and glycosidic bond hydrolysis in vivo. Such C3-DEA analogues would have much higher hypocholesterolemic activity. C3-DEA, and its methyl ester (C3-OMeDEA) and its methyl amido (C3-NMeDEA) were synthesized to examine their SAHH inhibitory and hypocholesterolemic activities. A crystal structure of SAHH containing C3-DEA was determined and confirmed that DEA and C3-DEA bound to the same site of SAHH with the same binding mode. The SAHH inhibitory activities of C3-DEA ( K_I=1.5μM) and C3-OMeDEA ( K_I=1.5μM) are significantly lower than that of DEA ( K_I=30nM), while rats fed by C3-DEA and C3-OMeDEA decrease the total plasma cholesterol and phospholipids by 36–40% and 23%, respectively, which is similar to the level of reductions (42% and 27%) by DEA. C3-NMeDEA lost most of the SAHH inhibitory activity ( K_I=30μM) and dietary C3-NMeDEA does not decrease cholesterol and phospholipid in plasma but decreases the triacylglycerol level by 16%. DEA and C3-DEA analogues are neither substrates nor inhibitors of adenosine deaminase.

Keywords: Abbreviations; ADA; adenosine deaminase; Ado; adenosine; C3-Ado; 3-deaza-adenosine; C3-DEA; 3-deaza-DEA; C3-NMeDEA; 3-deaza-DEA methylamide; C3-OMeDEA; 3-deaza-DEA methylester; D244E[NADH; +; Ado; ^*; ]; NADH and 3′-keto-adenosine (Ado*) bound rat liver D244E mutated SAHH; Hcy; Homocysteine; DEA; d; -eritadenine; NepA; neplanocin A; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PEG; poly(ethylene glycol); rmsd; root-mean-square deviation; SAH; S; -adenosyl-; l; -homocysteine; SAHH; S; -adenosyl-; l; -homocysteine hydrolase; SAHH[NAD; ⁺; +; DEA]; NAD; ⁺; and DEA bound rat liver SAHH; SAHH[NAD; ⁺; +; C3-DEA]; NAD; ⁺; and C3-DEA bound rat liver SAHH; SAM; S; -adenosyl-; l; -methionineEritadenine; 3-Deaza analogue; S; -Adenosylhomocysteine hydrolase; Enzyme inhibition; Cholesterol reduction; Chemical synthesis

Contribution of reactivated RUNX3 to inhibition of gastric cancer cell growth following suberoylanilide hydroxamic acid (vorinostat) treatment by Canhua Huang; Hiroshi Ida; Kosei Ito; Haiyuan Zhang; Yoshiaki Ito (pp. 990-1000).

Vorinostat (suberoylanilide hydroxamic acid, SAHA) represents a new class of highly potent histone deacetylase (HDAC) inhibitors that cause growth arrest, differentiation, and apoptosis of many tumor types in vitro and in vivo. RUNX3, a gastric tumor suppressor, is epigenetically silenced in gastric cancer cells. This study investigates the role of RUNX3 in vorinostat-induced suppression of gastric cancer cell growth. RUNX3 was up-regulated by vorinostat in gastric cancer cell lines not expressing RUNX3. In terms of cell viability, the mean IC₅₀ of vorinostat in RUNX3-negative cells was significantly lower than that seen in RUNX3-positive cells, indicating that the former are more sensitive to vorinostat in terms of growth arrest than are RUNX3-positive lines. The mechanism underlying this difference was found to be reactivation of RUNX3 expression by vorinostat and concomitant increase in acetylated histone H3 in the promoter region of RUNX3. Using three RUNX3-negative cell lines, we determined the contribution of RUNX3 reactivation to growth inhibition and induction of apoptosis following treatment of cells with vorinostat and found that up-regulated RUNX3 was significantly responsible for tumor suppressive activities.

Keywords: Abbreviations; SAHA; suberoylanilide hydroxamic acid; HDAC; histone deacetylase; HAT; histone acetylase; TSA; trichostatin A; ChIP; chromatin immunoprecipitation; MBD; methyl-CpG binding domain; DNMT; DNA methyltransferase; 5-aza-CdR; 5-aza-2′-deoxycytidineVorinostat; RUNX3; Gastric cancer; Chemotherapy

Matrix metalloproteinase-7-catalyzed release of HB-EGF mediates deoxycholyltaurine-induced proliferation of a human colon cancer cell line by Kunrong Cheng; Guofeng Xie; Jean-Pierre Raufman (pp. 1001-1012).

Prior evidence indicates that bile acids stimulate colon cancer cell proliferation by muscarinic receptor-induced transactivation of epidermal growth factor receptors (EGFR). To explore further the mechanism underlying this action, we tested the hypothesis that bile acids activate a matrix metalloproteinase (MMP) that catalyzes release of an EGFR ligand. Initial studies showed that non-selective MMP inhibitors blocked the actions of deoxycholyltaurine (DCT), thereby indicating a role for MMP-catalyzed release of an EGFR ligand. DCT-induced cell proliferation was reduced by increasing concentrations of EGFR kinase inhibitors, by antibodies to the ligand binding domain of EGFR, by neutralizing antibodies to heparin binding-EGF-like growth factor (HB-EGF) and by CRM197, an inhibitor of HB-EGF release. These findings and our observations with more selective MMP inhibitors suggested that MMP-7, an enzyme known to release HB-EGF, plays a key role in mediating bile acid-induced H508 colon cancer cell proliferation. We observed that recombinant HB-EGF and MMP-7 mimicked both the signaling and proliferative actions of bile acids. Strikingly, reducing MMP-7 expression with either neutralizing antibody or small interfering RNA attenuated the actions of DCT. MMP-7 expression in H508 cells was confirmed using quantitative reverse transcription PCR. DCT stimulated a greater than 10-fold increase in MMP-7 gene transcription. Co-localization of pro-MMP-7 and pro-HB-EGF at the cell surface (immunofluorescence microscopy) was demonstrated, indicating proximity of the enzyme to its substrate. These findings provide strong evidence that in H508 human colon cancer cells, DCT-induced transactivation of EGFR is mediated by MMP-7-catalyzed release of the EGFR ligand HB-EGF.

Keywords: Abbreviations; GPCR; G-protein coupled receptor; M; ₃; R; M; ₃; muscarinic receptor; ACh; acetylcholine; RTK; receptor tyrosine kinase; EGFR; epidermal growth factor receptor; TGF-α; transforming growth factor-α; MAPK; mitogen activated protein kinase; p90RSK; p90 ribosomal S kinase; HB-EGF; heparin binding-epidermal growth factor-like growth factor; MMP; matrix metalloproteinase; siRNA; small interfering RNA; DCT; deoxycholyltaurine; ADAM; a disintegrin and metalloproteaseMatrix metalloproteinases; Colon cancer cell proliferation; Bile acids; Epidermal growth factor receptors

The acetaminophen-derived bioactive N-acylphenolamine AM404 inhibits NFAT by targeting nuclear regulatory events by Francisco J. Caballero; Carmen M. Navarrete; Sandra Hess; Bernd L. Fiebich; Giovanni Appendino; Antonio Macho; Eduardo Muñoz; Rocío Sancho (pp. 1013-1023).

AM404 is a synthetic TRPV1/CB₁ hybrid ligand with inhibitory activity on the anandamide transporter and is used for the pharmacological manipulation of the endocannabinoid system. It has been recently described that acetaminophen is metabolised in the brain to form the bioactive N-acylphenolamine AM404 and therefore, we have evaluated the effect of this metabolite in human T cells, discovering that AM404 is a potent inhibitor of TCR-mediated T-cell activation. Moreover, we found that AM404 specifically inhibited both IL-2 and TNF-α gene transcription and TNF-α synthesis in CD3/CD28-stimulated Jurkat T cells in a FAAH independent way. To further characterize the biochemical inhibitory mechanisms of AM404, we examined the signaling pathways that regulate the activation of the transcription factors NF-κB, NFAT and AP-1 in Jurkat cells. We found that AM404 inhibited both the binding to DNA and the transcriptional activity of endogenous NFAT and the transcriptional activity driven by the over expressed fusion protein Gal4–NFAT (1–415). However, AM404 did not affect early steps in NFAT signaling such as CD3-induced calcium mobilization and NFAT1 dephosphorylation. The NFAT inhibitory activity of AM404 seems to be quite specific since this compound did not interfere with the signaling pathways leading to AP-1 or NF-κB activation. These findings provide new mechanistic insights into the immunological effects of AM404 which in part could explain some of the activities ascribed to the widely used acetaminophen.

Keywords: Abbreviations; AEA; anandamide; CB; cannabinoid receptor; COX; cyclooxygenase; FAAH; fatty acid amide hydrolase; JNK; Jun N terminal kinase; NADA; N; -arachydonoildopamine; NFAT; nuclear factor of activated T cells; NF-κB; nuclear factor of; kappa; B; PPAR; peroxisome proliferator-activated receptor; TRPV-1; transient release potential vanilloid receptor type 1T cells; Lipid mediators; Signal transduction

Curcumin induces the degradation of cyclin E expression through ubiquitin-dependent pathway and up-regulates cyclin-dependent kinase inhibitors p21 and p27 in multiple human tumor cell lines by Bharat B. Aggarwal; Sanjeev Banerjee; Uddalak Bharadwaj; Bokyung Sung; Shishir Shishodia; Gautam Sethi (pp. 1024-1032).

Curcumin, a well-known chemopreventive agent, has been shown to suppress the proliferation of a wide variety of tumor cells through a mechanism that is not fully understood. Cyclin E, a proto-oncogene that is overexpressed in many human cancers, mediates the G₁ to S transition, is a potential target of curcumin. We demonstrate in this report a dose- and time-dependent down-regulation of expression of cyclin E by curcumin that correlates with the decrease in the proliferation of human prostate and breast cancer cells. The suppression of cyclin E expression was not cell type dependent as down-regulation occurred in estrogen-positive and -negative breast cancer cells, androgen-dependent and -independent prostate cancer cells, leukemia and lymphoma cells, head and neck carcinoma cells, and lung cancer cells. Curcumin-induced down-regulation of cyclin E was reversed by proteasome inhibitors, lactacystin and N-acetyl-l-leucyl-l-leucyl-l-norleucinal, suggesting the role of ubiquitin-dependent proteasomal pathway. We found that curcumin enhanced the expression of tumor cyclin-dependent kinase (CDK) inhibitors p21 and p27 as well as tumor suppressor protein p53 but suppressed the expression of retinoblastoma protein. Curcumin also induced the accumulation of the cells in G1 phase of the cell cycle. Overall, our results suggest that proteasome-mediated down-regulation of cyclin E and up-regulation of CDK inhibitors may contribute to the antiproliferative effects of curcumin against various tumors.

Keywords: Abbreviations; CDK; cyclin-dependent kinase; ALLN; N; -acetyl-; l; -leucyl-; l; -leucyl-; l; -norleucinal; Rb; retinoblastoma; FBS; fetal bovine serumCurcumin; Cyclin E; Proteasome; p21; p27; p53

Relaxin receptors in hepatic stellate cells and cirrhotic liver by Robert G. Bennett; Shana R. Dalton; Katrina J. Mahan; Martha J. Gentry-Nielsen; Frederick G. Hamel; Dean J. Tuma (pp. 1033-1040).

The polypeptide hormone relaxin has antifibrotic effects on a number of tissues, including the liver. Central to the progression of hepatic fibrosis is the transdifferentiation of hepatic stellate cells (HSC) from a quiescent state to an activated, myofibroblastic phenotype that secretes fibrillar collagen. Relaxin inhibits markers of HSC activation, but relaxin receptor expression in the liver is unclear. The purpose of this study was to determine the expression of the relaxin receptors LGR7 and LGR8 in activated HSC. Production of cAMP was induced by treatment of HSC with relaxin, or the relaxin-related peptides InsL3 or relaxin-3, selective activators of LGR8 and LGR7, respectively. Quiescent HSC expressed low levels of LGR7 but not LGR8. During progression to the activated phenotype, expression of both receptors increased markedly. Immunocytochemistry confirmed the presence of both receptors in activated HSC. In normal rat liver, LGR7, but not LGR8, was expressed at low levels. In cirrhotic liver, expression of both receptors significantly increased. Neither receptor was detectable in normal liver by immunohistochemistry, but both LGR7 and LGR8 were readily detectable in cirrhosis. These results were confirmed in human cirrhotic tissue, with the additional finding of occasional perisinusoidal LGR7 immunoreactivity in non-cirrhotic tissue. In conclusion, the expression of LGR7 and LGR8 is increased with activation of HSC in culture. Cirrhosis also caused increased expression of both receptors. Therefore, agents that stimulate LGR8 and LGR7 may be therapeutically useful to limit the activation of hepatic stellate cells in liver injury.

Keywords: Abbreviations; HSC; hepatic stellate cells; SMA; smooth muscle α-actin; InsL; insulin-like peptide; LGR; leucine-rich G-protein-coupled receptorFibrosis; LGR7; LGR8; InsL3; Relaxin-3

Sections

Personal tools

Biochemical Pharmacology (v.73, #7)

Sections

Personal tools

Local resources

Biochemical Pharmacology (v.73, #7)