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Biochemical Pharmacology (v.84, #12)
Nous Sommes Tous des Bacteries: Implications for medicine, pharmacology and public health by David J. Triggle (pp. 1543-1550).
As a species we humans are outnumbered by bacteria in both cell and gene count. This somewhat humbling observation is key to the increasing recognition that the long-standing symbiotic and commensal relations between Homo sapiens and bacteria are of great significance to basic human physiology and health. Knowledge of our human bacterial environment is contributing to an understanding of a variety of disorders including obesity and metabolic syndrome, cardiovascular disease, immunity, and neuronal development and behavior. The Human Microbiome Project is providing a genetic and ecological analysis and will serve as a parallel to the Human Genome Project. Exploration of the chemical space utilized by bacteria will contribute to the development of new small molecule therapeutic agents, including new antibiotics. And genetically re-engineered bacteria are proving to be of potential value as actual therapeutic entities. Our understanding of our bacterial world has the capability to transform radically our current approach to human health diverting it from an emphasis on acute treatments to living in healthy harmony with both our internal and external environments.
Keywords: Bacteria; Obesity; Cancer; Diabetes; Neurobiology; Microbiome; Commensal; Symbionts
Nous Sommes Tous des Bacteries: Implications for medicine, pharmacology and public health by David J. Triggle (pp. 1543-1550).
As a species we humans are outnumbered by bacteria in both cell and gene count. This somewhat humbling observation is key to the increasing recognition that the long-standing symbiotic and commensal relations between Homo sapiens and bacteria are of great significance to basic human physiology and health. Knowledge of our human bacterial environment is contributing to an understanding of a variety of disorders including obesity and metabolic syndrome, cardiovascular disease, immunity, and neuronal development and behavior. The Human Microbiome Project is providing a genetic and ecological analysis and will serve as a parallel to the Human Genome Project. Exploration of the chemical space utilized by bacteria will contribute to the development of new small molecule therapeutic agents, including new antibiotics. And genetically re-engineered bacteria are proving to be of potential value as actual therapeutic entities. Our understanding of our bacterial world has the capability to transform radically our current approach to human health diverting it from an emphasis on acute treatments to living in healthy harmony with both our internal and external environments.
Keywords: Bacteria; Obesity; Cancer; Diabetes; Neurobiology; Microbiome; Commensal; Symbionts
Sphingosine-1-phosphate receptors as emerging targets for treatment of pain by Sandra P. Welch; Laura J. Sim-Selley; Dana E. Selley (pp. 1551-1562).
Lysolipids are important mediators of cellular communication in multiple physiological processes. Sphingosine-1-phosphate (S1P) is a major lysolipid in many organs, including the central nervous system (CNS). This commentary discusses recent findings on the role of S1P in regulating pain perception, and highlights advances and challenges in the field. S1P interacts with multiple cellular targets, including G-protein-coupled receptors. Known S1P receptors include five types, four of which are expressed in the CNS (S1P1,2,3,5) where they are localized on neurons and glia. S1P receptor-mediated G-protein activation has been demonstrated throughout the CNS, including regions that regulate nociception. S1P receptors couple to multiple G-proteins to produce various intracellular responses, and can mediate both excitatory and inhibitory neuromodulation, depending on the receptor type and cellular context. Both antinociceptive and pro-nociceptive effects of S1P have been reported, and both actions can involve S1P1 receptors. Current evidence suggests that antinociception is mediated by CNS neurons, whereas pro-nociception is mediated by primary afferent neurons or immune cells in the periphery, or CNS glia. Nonetheless, peripheral administration of the S1P1,3,4,5 agonist pro-drug, FTY720, produces antinociception. FTY720 is approved to treat multiple sclerosis, and produces potent anti-inflammatory effects, which suggests potential utility for painful autoimmune diseases. Furthermore, evidence suggests that the S1P system interacts with other pain-modulatory systems, such as endogenous cannabinoid and opioid systems, and putative novel sphingolipid targets in the CNS. These findings suggest that drugs targeting the S1P system could be developed as novel analgesics, either as monotherapy or potential adjuncts to established analgesics.
Keywords: Sphingosine-1-phosphate; S1P receptors; Antinociception; Hyperalgesia; FTY720; Sphingolipids
Sphingosine-1-phosphate receptors as emerging targets for treatment of pain by Sandra P. Welch; Laura J. Sim-Selley; Dana E. Selley (pp. 1551-1562).
Lysolipids are important mediators of cellular communication in multiple physiological processes. Sphingosine-1-phosphate (S1P) is a major lysolipid in many organs, including the central nervous system (CNS). This commentary discusses recent findings on the role of S1P in regulating pain perception, and highlights advances and challenges in the field. S1P interacts with multiple cellular targets, including G-protein-coupled receptors. Known S1P receptors include five types, four of which are expressed in the CNS (S1P1,2,3,5) where they are localized on neurons and glia. S1P receptor-mediated G-protein activation has been demonstrated throughout the CNS, including regions that regulate nociception. S1P receptors couple to multiple G-proteins to produce various intracellular responses, and can mediate both excitatory and inhibitory neuromodulation, depending on the receptor type and cellular context. Both antinociceptive and pro-nociceptive effects of S1P have been reported, and both actions can involve S1P1 receptors. Current evidence suggests that antinociception is mediated by CNS neurons, whereas pro-nociception is mediated by primary afferent neurons or immune cells in the periphery, or CNS glia. Nonetheless, peripheral administration of the S1P1,3,4,5 agonist pro-drug, FTY720, produces antinociception. FTY720 is approved to treat multiple sclerosis, and produces potent anti-inflammatory effects, which suggests potential utility for painful autoimmune diseases. Furthermore, evidence suggests that the S1P system interacts with other pain-modulatory systems, such as endogenous cannabinoid and opioid systems, and putative novel sphingolipid targets in the CNS. These findings suggest that drugs targeting the S1P system could be developed as novel analgesics, either as monotherapy or potential adjuncts to established analgesics.
Keywords: Sphingosine-1-phosphate; S1P receptors; Antinociception; Hyperalgesia; FTY720; Sphingolipids
Hypoxia, Mn-SOD and H2O2 regulate p53 reactivation and PRIMA-1 toxicity irrespective of p53 status in human breast cancer cells by Manuel Rieber; Mary Strasberg-Rieber (pp. 1563-1570).
Hypoxia is part of the tumor microenvironment favoring cancer resistance to chemotherapy mediated by mutations in the tumor suppressor p53 gene (TP53), or by conformational wt TP53 dysfunction. Since it is important to suppress tumor adaptation to hypoxia, irrespective of p53 status, we compared the efficacy of nutlin-3 which prevents MDM2-wt p53 interactions and PRIMA-1 which promotes mutant p53 reactivation and induction of massive apoptosis, under normoxia and hypoxia, against (a) SKBR3 breast carcinoma harboring a mutant p53R175H and over-expressing erbB2; and (b) genetically matched breast cancer ERα positive MCF-7 cells harboring either wt p53 or mutant p53 R175H. Under normoxia, PRIMA-1 was active against breast cancer cells harboring mutant p53. However, hypoxia further increased the susceptibility of mutant p53 breast cancer SKBR3 cells to lower PRIMA-1 levels, possibly through oxidative stress since this was counteracted by N-acetylcysteine. When using MCF-7 cells over-expressing mutant p53, PRIMA-1 synergized with exogenous peroxidase to increase apoptosis concomitantly with induction of PUMA and Mn-SOD, under normoxia. Wt p53 MCF-7 cells responded to hypoxia by increasing superoxide dismutase and their reactivity with the PAb240 antibody, known to recognize conformationally-inactive p53. This correlated with sensitization of wt p53 MCF-7 cells to PRIMA-1 but not to nutlin-3. PRIMA-1 toxicity against normoxic wt p53 MCF-7 cells was also decreased by Mn-SOD over-expression or when added with the glutathione antagonist, buthionine sulfoximine. This report shows for the first time that hypoxia increases PRIMA-1 toxicity in human breast cancer cells, partly by modulating p53 conformation and by inducing superoxide turnover. Our results suggest that PRIMA-1 may help to prevent hypoxia-mediated tumor chemoresistance.
Keywords: Abbreviations; DN-mutant p53 R175H; dominant-negative mutant p53 Arg 175His; wt; wild type; PARP; poly(ADP-ribose) polymerase; [Mn-SOD, SOD2]; manganese-dependent superoxide dismutase; [Cu/Zn-SOD, SOD1]; copper–zinc-dependent superoxide dismutase; BSO; buthionine sulfoximine; ROS; reactive oxygen species; NAC; N-acetylcysteine; GSH; glutathioneNutlin; Superoxide dismutase; Hypoxia; p53 reactivation; Oxidative stress; PRIMA-1
Hypoxia, Mn-SOD and H2O2 regulate p53 reactivation and PRIMA-1 toxicity irrespective of p53 status in human breast cancer cells by Manuel Rieber; Mary Strasberg-Rieber (pp. 1563-1570).
Hypoxia is part of the tumor microenvironment favoring cancer resistance to chemotherapy mediated by mutations in the tumor suppressor p53 gene (TP53), or by conformational wt TP53 dysfunction. Since it is important to suppress tumor adaptation to hypoxia, irrespective of p53 status, we compared the efficacy of nutlin-3 which prevents MDM2-wt p53 interactions and PRIMA-1 which promotes mutant p53 reactivation and induction of massive apoptosis, under normoxia and hypoxia, against (a) SKBR3 breast carcinoma harboring a mutant p53R175H and over-expressing erbB2; and (b) genetically matched breast cancer ERα positive MCF-7 cells harboring either wt p53 or mutant p53 R175H. Under normoxia, PRIMA-1 was active against breast cancer cells harboring mutant p53. However, hypoxia further increased the susceptibility of mutant p53 breast cancer SKBR3 cells to lower PRIMA-1 levels, possibly through oxidative stress since this was counteracted by N-acetylcysteine. When using MCF-7 cells over-expressing mutant p53, PRIMA-1 synergized with exogenous peroxidase to increase apoptosis concomitantly with induction of PUMA and Mn-SOD, under normoxia. Wt p53 MCF-7 cells responded to hypoxia by increasing superoxide dismutase and their reactivity with the PAb240 antibody, known to recognize conformationally-inactive p53. This correlated with sensitization of wt p53 MCF-7 cells to PRIMA-1 but not to nutlin-3. PRIMA-1 toxicity against normoxic wt p53 MCF-7 cells was also decreased by Mn-SOD over-expression or when added with the glutathione antagonist, buthionine sulfoximine. This report shows for the first time that hypoxia increases PRIMA-1 toxicity in human breast cancer cells, partly by modulating p53 conformation and by inducing superoxide turnover. Our results suggest that PRIMA-1 may help to prevent hypoxia-mediated tumor chemoresistance.
Keywords: Abbreviations; DN-mutant p53 R175H; dominant-negative mutant p53 Arg 175His; wt; wild type; PARP; poly(ADP-ribose) polymerase; [Mn-SOD, SOD2]; manganese-dependent superoxide dismutase; [Cu/Zn-SOD, SOD1]; copper–zinc-dependent superoxide dismutase; BSO; buthionine sulfoximine; ROS; reactive oxygen species; NAC; N-acetylcysteine; GSH; glutathioneNutlin; Superoxide dismutase; Hypoxia; p53 reactivation; Oxidative stress; PRIMA-1
Apigenin induces DNA damage through the PKCδ-dependent activation of ATM and H2AX causing down-regulation of genes involved in cell cycle control and DNA repair by Daniel Arango; Arti Parihar; Frederick A. Villamena; Liwen Wang; Michael A. Freitas; Erich Grotewold; Andrea I. Doseff (pp. 1571-1580).
Apigenin, an abundant plant flavonoid, exhibits anti-proliferative and anti-carcinogenic activities through mechanisms yet not fully defined. In the present study, we show that the treatment of leukemia cells with apigenin resulted in the induction of DNA damage preceding the activation of the apoptotic program. Apigenin-induced DNA damage was mediated by p38 and protein kinase C-delta (PKCδ), yet was independent of reactive oxygen species or caspase activity. Treatment of monocytic leukemia cells with apigenin induced the phosphorylation of the ataxia-telangiectasia mutated (ATM) kinase and histone H2AX, two key regulators of the DNA damage response, without affecting the ataxia-telangiectasia mutated and Rad-3-related (ATR) kinase. Silencing and pharmacological inhibition of PKCδ abrogated ATM and H2AX phosphorylation, whereas inhibition of p38 reduced H2AX phosphorylation independently of ATM. We established that apigenin delayed cell cycle progression at G1/S and increased the number of apoptotic cells. In addition, genome-wide mRNA analyses showed that apigenin-induced DNA damage led to down-regulation of genes involved in cell-cycle control and DNA repair. Taken together, the present results show that the PKCδ-dependent activation of ATM and H2AX define the signaling networks responsible for the regulation of DNA damage promoting genome-wide mRNA alterations that result in cell cycle arrest, hence contributing to the anti-carcinogenic activities of this flavonoid.
Keywords: Flavonoids; Apoptosis; DNA damage; PKC delta
Apigenin induces DNA damage through the PKCδ-dependent activation of ATM and H2AX causing down-regulation of genes involved in cell cycle control and DNA repair by Daniel Arango; Arti Parihar; Frederick A. Villamena; Liwen Wang; Michael A. Freitas; Erich Grotewold; Andrea I. Doseff (pp. 1571-1580).
Apigenin, an abundant plant flavonoid, exhibits anti-proliferative and anti-carcinogenic activities through mechanisms yet not fully defined. In the present study, we show that the treatment of leukemia cells with apigenin resulted in the induction of DNA damage preceding the activation of the apoptotic program. Apigenin-induced DNA damage was mediated by p38 and protein kinase C-delta (PKCδ), yet was independent of reactive oxygen species or caspase activity. Treatment of monocytic leukemia cells with apigenin induced the phosphorylation of the ataxia-telangiectasia mutated (ATM) kinase and histone H2AX, two key regulators of the DNA damage response, without affecting the ataxia-telangiectasia mutated and Rad-3-related (ATR) kinase. Silencing and pharmacological inhibition of PKCδ abrogated ATM and H2AX phosphorylation, whereas inhibition of p38 reduced H2AX phosphorylation independently of ATM. We established that apigenin delayed cell cycle progression at G1/S and increased the number of apoptotic cells. In addition, genome-wide mRNA analyses showed that apigenin-induced DNA damage led to down-regulation of genes involved in cell-cycle control and DNA repair. Taken together, the present results show that the PKCδ-dependent activation of ATM and H2AX define the signaling networks responsible for the regulation of DNA damage promoting genome-wide mRNA alterations that result in cell cycle arrest, hence contributing to the anti-carcinogenic activities of this flavonoid.
Keywords: Flavonoids; Apoptosis; DNA damage; PKC delta
Identification of novel Sp1 targets involved in proliferation and cancer by functional genomics by Carlota Oleaga; Sabine Welten; Audrey Belloc; Anna Solé; Laura Rodriguez; Núria Mencia; Elisabet Selga; Alicia Tapias; Veronique Noé; Carlos J. Ciudad (pp. 1581-1591).
Sp1 is a transcription factor regulating many genes through its DNA binding domain, containing three zinc fingers. We were interested in identifying target genes regulated by Sp1, particularly those involved in proliferation and cancer. Our approach was to treat HeLa cells with a siRNA directed against Sp1 mRNA to decrease the expression of Sp1 and, in turn, the genes activated by this transcription factor. Sp1-siRNA treatment led to a great number of differentially expressed genes as determined by whole genome cDNA microarray analysis. Underexpressed genes were selected since they represent putative genes activated by Sp1 and classified in six Gene Onthology categories, namely proliferation and cancer, mRNA processing, lipid metabolism, glucidic metabolism, transcription and translation. Putative Sp1 binding sites were found in the promoters of the selected genes using the MatchTM software. After literature mining, 11 genes were selected for further validation. Underexpression by qRT-PCR was confirmed for the 11 genes plus Sp1 in HeLa cells after Sp1-siRNA treatment. EMSA and ChIP assays were performed to test for binding of Sp1 to the promoters of these genes. We observed binding of Sp1 to the promoters of RAB20, FGF21, IHPK2, ARHGAP18, NPM3, SRSF7, CALM3, PGD and Sp1 itself. Furthermore, the mRNA levels of RAB20, FGF21 and IHPK2 and luciferase activity for these three genes related to proliferation and cancer, were determined after overexpression of Sp1 in HeLa cells, to confirm their regulation by Sp1. Involvement of these three genes in proliferation was validated by gene silencing using polypurine reverse hoogsteen hairpins.
Keywords: Sp1; Target; Genomics; Proliferation; CancerAbbreviations; FDR; false discovery rate; GEO; gene expression omnibus; GO; gene ontology; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NE; nuclear extract; NP-40; nonyl phenoxypolyethoxylethanol; PPRHs; polypurine reverse hoogsteen hairpins; RMA; robust multichip average; siNR; siRNA non related; siSp1; siRNA directed against Sp1; TF; transcription factor
Identification of novel Sp1 targets involved in proliferation and cancer by functional genomics by Carlota Oleaga; Sabine Welten; Audrey Belloc; Anna Solé; Laura Rodriguez; Núria Mencia; Elisabet Selga; Alicia Tapias; Veronique Noé; Carlos J. Ciudad (pp. 1581-1591).
Sp1 is a transcription factor regulating many genes through its DNA binding domain, containing three zinc fingers. We were interested in identifying target genes regulated by Sp1, particularly those involved in proliferation and cancer. Our approach was to treat HeLa cells with a siRNA directed against Sp1 mRNA to decrease the expression of Sp1 and, in turn, the genes activated by this transcription factor. Sp1-siRNA treatment led to a great number of differentially expressed genes as determined by whole genome cDNA microarray analysis. Underexpressed genes were selected since they represent putative genes activated by Sp1 and classified in six Gene Onthology categories, namely proliferation and cancer, mRNA processing, lipid metabolism, glucidic metabolism, transcription and translation. Putative Sp1 binding sites were found in the promoters of the selected genes using the MatchTM software. After literature mining, 11 genes were selected for further validation. Underexpression by qRT-PCR was confirmed for the 11 genes plus Sp1 in HeLa cells after Sp1-siRNA treatment. EMSA and ChIP assays were performed to test for binding of Sp1 to the promoters of these genes. We observed binding of Sp1 to the promoters of RAB20, FGF21, IHPK2, ARHGAP18, NPM3, SRSF7, CALM3, PGD and Sp1 itself. Furthermore, the mRNA levels of RAB20, FGF21 and IHPK2 and luciferase activity for these three genes related to proliferation and cancer, were determined after overexpression of Sp1 in HeLa cells, to confirm their regulation by Sp1. Involvement of these three genes in proliferation was validated by gene silencing using polypurine reverse hoogsteen hairpins.
Keywords: Sp1; Target; Genomics; Proliferation; CancerAbbreviations; FDR; false discovery rate; GEO; gene expression omnibus; GO; gene ontology; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NE; nuclear extract; NP-40; nonyl phenoxypolyethoxylethanol; PPRHs; polypurine reverse hoogsteen hairpins; RMA; robust multichip average; siNR; siRNA non related; siSp1; siRNA directed against Sp1; TF; transcription factor
Knockdown of stromal interaction molecule 1 (STIM1) suppresses store-operated calcium entry, cell proliferation and tumorigenicity in human epidermoid carcinoma A431 cells by Junko Yoshida; Kuniyoshi Iwabuchi; Tadashi Matsui; Takaharu Ishibashi; Takayoshi Masuoka; Matomo Nishio (pp. 1592-1603).
Store-operated calcium (Ca2+) entry (SOCE) is important for cellular activities such as gene transcription, cell cycle progression and proliferation in most non-excitable cells. Stromal interaction molecule 1 (STIM1), a newly identified Ca2+-sensing protein, monitors the depletion of endoplasmic reticulum (ER) Ca2+ stores and activates store-operated Ca2+ channels at the plasma membrane to induce SOCE. To investigate the possible roles of STIM1 in tumor growth in relation to SOCE, we established STIM1 knockdown (KD) clones of human epidermoid carcinoma A431 cells by RNA interference. Thapsigargin, an inhibitor of ER Ca2+-ATPase, -induced and phospholipase C-coupled receptor agonist-induced SOCEs were reduced in two STIM1 KD clones compared to a negative control clone. Re-expression of a KD-resistant full-length STIM1, but not a Ca2+ release-activated Ca2+ channel activation domain (CAD)-deleted STIM1 mutant, in the KD clone restored the amplitude of SOCE, suggesting the specificity of the STIM1 knockdown. The cell growth of the STIM1 KD clones was slower than that of the negative control clone. DNA synthesis assessed by BrdU incorporation, as well as EGF-stimulated EGF receptor activation, decreased in the STIM1 KD clones. Xenograft growth of the STIM1 KD clones was significantly retarded compared with that of the negative control. Cell migration was attenuated in the STIM1 KD clone and the STIM1 silencing effect was reversed by transient re-expression of the full-length STIM1 but not CAD-deletion mutant. These results indicate that STIM1 plays an important role in SOCE, cell-growth and tumorigenicity in human epidermoid carcinoma A431cells, suggesting the potential use of STIM1-targeting agents for treating epidermoid carcinoma.
Keywords: Stromal interaction molecule 1 (STIM1); Store-operated calcium entry; Proliferation; Tumorigenicity; RNA interference; Human epidermoid carcinoma A431 cells
Knockdown of stromal interaction molecule 1 (STIM1) suppresses store-operated calcium entry, cell proliferation and tumorigenicity in human epidermoid carcinoma A431 cells by Junko Yoshida; Kuniyoshi Iwabuchi; Tadashi Matsui; Takaharu Ishibashi; Takayoshi Masuoka; Matomo Nishio (pp. 1592-1603).
Store-operated calcium (Ca2+) entry (SOCE) is important for cellular activities such as gene transcription, cell cycle progression and proliferation in most non-excitable cells. Stromal interaction molecule 1 (STIM1), a newly identified Ca2+-sensing protein, monitors the depletion of endoplasmic reticulum (ER) Ca2+ stores and activates store-operated Ca2+ channels at the plasma membrane to induce SOCE. To investigate the possible roles of STIM1 in tumor growth in relation to SOCE, we established STIM1 knockdown (KD) clones of human epidermoid carcinoma A431 cells by RNA interference. Thapsigargin, an inhibitor of ER Ca2+-ATPase, -induced and phospholipase C-coupled receptor agonist-induced SOCEs were reduced in two STIM1 KD clones compared to a negative control clone. Re-expression of a KD-resistant full-length STIM1, but not a Ca2+ release-activated Ca2+ channel activation domain (CAD)-deleted STIM1 mutant, in the KD clone restored the amplitude of SOCE, suggesting the specificity of the STIM1 knockdown. The cell growth of the STIM1 KD clones was slower than that of the negative control clone. DNA synthesis assessed by BrdU incorporation, as well as EGF-stimulated EGF receptor activation, decreased in the STIM1 KD clones. Xenograft growth of the STIM1 KD clones was significantly retarded compared with that of the negative control. Cell migration was attenuated in the STIM1 KD clone and the STIM1 silencing effect was reversed by transient re-expression of the full-length STIM1 but not CAD-deletion mutant. These results indicate that STIM1 plays an important role in SOCE, cell-growth and tumorigenicity in human epidermoid carcinoma A431cells, suggesting the potential use of STIM1-targeting agents for treating epidermoid carcinoma.
Keywords: Stromal interaction molecule 1 (STIM1); Store-operated calcium entry; Proliferation; Tumorigenicity; RNA interference; Human epidermoid carcinoma A431 cells
2-Deoxy-d-glucose cooperates with arsenic trioxide to induce apoptosis in leukemia cells: Involvement of IGF-1R-regulated Akt/mTOR, MEK/ERK and LKB-1/AMPK signaling pathways by María Cristina Estañ; Eva Calviño; Elena de Blas; María del Carmen Boyano-Adánez; Maria Luz Mena; Milagros Gómez-Gómez; Eduardo Rial; Patricio Aller (pp. 1604-1616).
2-Deoxy-d-Glucose (2-DG) causes mitochondrial dysfunction, IGF-1R-mediated activation of Akt and ERK defensive kinases, which is prevented by arsenic trioxide (ATO), and occasional inactivation of AMPK. These events facilitate the activation of the mitochondrial apoptotic pathway.While the anti-tumor efficacy of 2-deoxy-d-glucose (2-DG) is normally low in monotherapy, it may represent a valuable radio- and chemo-sensitizing agent. We here demonstrate that 2–10mM 2-DG cooperates with arsenic trioxide (ATO) and other antitumor drugs to induce apoptosis in human myeloid leukemia cell lines. Using ATO and HL60 as drug and cell models, respectively, we observed that 2-DG/ATO combination activates the mitochondrial apoptotic pathway, as indicated by Bid-, and Bax-regulated cytochrome c and Omi/HtrA2 release, XIAP down-regulation, and caspase-9/-3 pathway activation. 2-DG neither causes oxidative stress nor increases ATO uptake, but causes inner mitochondria membrane permeabilization as well as moderate ATP depletion, which nevertheless do not satisfactorily explain the pro-apoptotic response. Surprisingly 2-DG causes cell line-specific decrease in LKB-1/AMPK phosphorylation/activation, and also causes Akt/mTOR/p70S6K and MEK/ERK activation, which is prevented by co-treatment with ATO. The use of kinase-specific pharmacologic inhibitors and/or siRNAs reveals that apoptosis is facilitated by AMPK inactivation and restrained by Akt and ERK activation, and that Akt and ERK activation mediates AMPK inhibition. Finally, 2-DG stimulates IGF-1R phosphorylation/activation, and co-treatment with IGF-1R inhibitor prevents 2-DG effects on Akt, ERK and AMPK, and facilitates 2-DG-provoked apoptosis. In summary 2-DG elicits IGF-1R-mediated AMPK inactivation and Akt and ERK activation, which facilitates or restrain apoptosis, respectively. 2-DG-provoked AMPK inactivation increases the apoptotic efficacy of ATO, while in turn ATO-provoked Akt and ERK inactivation may increase the efficacy of 2-DG as anti-tumor drug.
Keywords: Abbreviations; AMPK; AMP-activated kinase; Akt; protein kinase B; Akt; i; V; Akt inhibitor V, triciribine; AMPK; AMP-activated kinase; AML; acute myeloid leukemia; APL; acute promyelocytic leukemia; ATO; arsenic trioxide; CC; AMPK inhibitor, Compound C; DAPI; 4,6-diamino-2-phenylindole; 2-DG; 2-deoxy-; d; -glucose; DHE; dihydroethidium; ERK; extracellular signal-regulated kinase; GSH; reduced glutathione; H; 2; DCFDA; dichlorodihydrofluorescein diacetate; IGF-1; insulin-like growth factor-1; IGF-1R; insulin-like growth factor-1 receptor; LKB-1; liver kinase B1; LY294002; 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; mAb; monoclonal antibody; mTOR; mammalian target of rapamycin; MAPK; mitogen-activated protein kinase; MEK; mitogen-induced extracellular kinase/extracellular signal-regulated kinase; mIMP; inner mitochondrial membrane permeabilization; mOMP; outer mitochondrial membrane permeabilization; mPTP; mitochondrial transition pore; MTT; 3(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide; PBLs; peripheral blood lymphocytes; PBS; phosphate buffered saline; PI3K; phosphatidylinositol 3-kinase; PI; propidium iodide; R123; rhodamine 123; PQ401; IGF-1R inhibitor II, N-(2-Methoxy-5-chlorophenyl)-; N; ′-(2methylquinilin-4-yl)-urea; TNF-α; tumor necrosis factor-α; U0126; 1,4-Diamino-2,3-dicyano-1,4-; bis; (2-aminophenylthio)butadiene; z-VAD-fmk; Z-Val-Ala-Asp(OMe)-CH; 2; F2-Deoxy-; d; -glucose; Arsenic trioxide; Apoptosis; Protein kinases; Leukemia cells
2-Deoxy-d-glucose cooperates with arsenic trioxide to induce apoptosis in leukemia cells: Involvement of IGF-1R-regulated Akt/mTOR, MEK/ERK and LKB-1/AMPK signaling pathways by María Cristina Estañ; Eva Calviño; Elena de Blas; María del Carmen Boyano-Adánez; Maria Luz Mena; Milagros Gómez-Gómez; Eduardo Rial; Patricio Aller (pp. 1604-1616).
2-Deoxy-d-Glucose (2-DG) causes mitochondrial dysfunction, IGF-1R-mediated activation of Akt and ERK defensive kinases, which is prevented by arsenic trioxide (ATO), and occasional inactivation of AMPK. These events facilitate the activation of the mitochondrial apoptotic pathway.While the anti-tumor efficacy of 2-deoxy-d-glucose (2-DG) is normally low in monotherapy, it may represent a valuable radio- and chemo-sensitizing agent. We here demonstrate that 2–10mM 2-DG cooperates with arsenic trioxide (ATO) and other antitumor drugs to induce apoptosis in human myeloid leukemia cell lines. Using ATO and HL60 as drug and cell models, respectively, we observed that 2-DG/ATO combination activates the mitochondrial apoptotic pathway, as indicated by Bid-, and Bax-regulated cytochrome c and Omi/HtrA2 release, XIAP down-regulation, and caspase-9/-3 pathway activation. 2-DG neither causes oxidative stress nor increases ATO uptake, but causes inner mitochondria membrane permeabilization as well as moderate ATP depletion, which nevertheless do not satisfactorily explain the pro-apoptotic response. Surprisingly 2-DG causes cell line-specific decrease in LKB-1/AMPK phosphorylation/activation, and also causes Akt/mTOR/p70S6K and MEK/ERK activation, which is prevented by co-treatment with ATO. The use of kinase-specific pharmacologic inhibitors and/or siRNAs reveals that apoptosis is facilitated by AMPK inactivation and restrained by Akt and ERK activation, and that Akt and ERK activation mediates AMPK inhibition. Finally, 2-DG stimulates IGF-1R phosphorylation/activation, and co-treatment with IGF-1R inhibitor prevents 2-DG effects on Akt, ERK and AMPK, and facilitates 2-DG-provoked apoptosis. In summary 2-DG elicits IGF-1R-mediated AMPK inactivation and Akt and ERK activation, which facilitates or restrain apoptosis, respectively. 2-DG-provoked AMPK inactivation increases the apoptotic efficacy of ATO, while in turn ATO-provoked Akt and ERK inactivation may increase the efficacy of 2-DG as anti-tumor drug.
Keywords: Abbreviations; AMPK; AMP-activated kinase; Akt; protein kinase B; Akt; i; V; Akt inhibitor V, triciribine; AMPK; AMP-activated kinase; AML; acute myeloid leukemia; APL; acute promyelocytic leukemia; ATO; arsenic trioxide; CC; AMPK inhibitor, Compound C; DAPI; 4,6-diamino-2-phenylindole; 2-DG; 2-deoxy-; d; -glucose; DHE; dihydroethidium; ERK; extracellular signal-regulated kinase; GSH; reduced glutathione; H; 2; DCFDA; dichlorodihydrofluorescein diacetate; IGF-1; insulin-like growth factor-1; IGF-1R; insulin-like growth factor-1 receptor; LKB-1; liver kinase B1; LY294002; 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; mAb; monoclonal antibody; mTOR; mammalian target of rapamycin; MAPK; mitogen-activated protein kinase; MEK; mitogen-induced extracellular kinase/extracellular signal-regulated kinase; mIMP; inner mitochondrial membrane permeabilization; mOMP; outer mitochondrial membrane permeabilization; mPTP; mitochondrial transition pore; MTT; 3(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide; PBLs; peripheral blood lymphocytes; PBS; phosphate buffered saline; PI3K; phosphatidylinositol 3-kinase; PI; propidium iodide; R123; rhodamine 123; PQ401; IGF-1R inhibitor II, N-(2-Methoxy-5-chlorophenyl)-; N; ′-(2methylquinilin-4-yl)-urea; TNF-α; tumor necrosis factor-α; U0126; 1,4-Diamino-2,3-dicyano-1,4-; bis; (2-aminophenylthio)butadiene; z-VAD-fmk; Z-Val-Ala-Asp(OMe)-CH; 2; F2-Deoxy-; d; -glucose; Arsenic trioxide; Apoptosis; Protein kinases; Leukemia cells
The anticancer multi-kinase inhibitor dovitinib also targets topoisomerase I and topoisomerase II by Brian B. Hasinoff; Xing Wu; John L. Nitiss; Ragu Kanagasabai; Jack C. Yalowich (pp. 1617-1626).
Dovitinib (TKI258/CHIR258) is a multi-kinase inhibitor in phase III development for the treatment of several cancers. Dovitinib is a benzimidazole-quinolinone compound that structurally resembles the bisbenzimidazole minor groove binding dye Hoechst 33258. Dovitinib bound to DNA as shown by its ability to increase the DNA melting temperature and by increases in its fluorescence spectrum that occurred upon the addition of DNA. Molecular modeling studies of the docking of dovitinib into an X-ray structure of a Hoechst 33258–DNA complex showed that dovitinib could reasonably be accommodated in the DNA minor groove. Because DNA binders are often topoisomerase I (EC 5.99.1.2) and topoisomerase II (EC 5.99.1.3) inhibitors, the ability of dovitinib to inhibit these DNA processing enzymes was also investigated. Dovitinib inhibited the catalytic decatenation activity of topoisomerase IIα. It also inhibited the DNA-independent ATPase activity of yeast topoisomerase II which suggested that it interacted with the ATP binding site. Using isolated human topoisomerase IIα, dovitinib stabilized the enzyme-cleavage complex and acted as a topoisomerase IIα poison. Dovitinib was also found to be a cellular topoisomerase II poison in human leukemia K562 cells and induced double-strand DNA breaks in K562 cells as evidenced by increased phosphorylation of H2AX. Finally, dovitinib inhibited the topoisomerase I-catalyzed relaxation of plasmid DNA and acted as a cellular topoisomerase I poison. In conclusion, the cell growth inhibitory activity and the anticancer activity of dovitinib may result not only from its ability to inhibit multiple kinases, but also, in part, from its ability to target topoisomerase I and topoisomerase II.
Keywords: Dovitinib; Anticancer; DNA; Topoisomerase II; Topoisomerase I
The anticancer multi-kinase inhibitor dovitinib also targets topoisomerase I and topoisomerase II by Brian B. Hasinoff; Xing Wu; John L. Nitiss; Ragu Kanagasabai; Jack C. Yalowich (pp. 1617-1626).
Dovitinib (TKI258/CHIR258) is a multi-kinase inhibitor in phase III development for the treatment of several cancers. Dovitinib is a benzimidazole-quinolinone compound that structurally resembles the bisbenzimidazole minor groove binding dye Hoechst 33258. Dovitinib bound to DNA as shown by its ability to increase the DNA melting temperature and by increases in its fluorescence spectrum that occurred upon the addition of DNA. Molecular modeling studies of the docking of dovitinib into an X-ray structure of a Hoechst 33258–DNA complex showed that dovitinib could reasonably be accommodated in the DNA minor groove. Because DNA binders are often topoisomerase I (EC 5.99.1.2) and topoisomerase II (EC 5.99.1.3) inhibitors, the ability of dovitinib to inhibit these DNA processing enzymes was also investigated. Dovitinib inhibited the catalytic decatenation activity of topoisomerase IIα. It also inhibited the DNA-independent ATPase activity of yeast topoisomerase II which suggested that it interacted with the ATP binding site. Using isolated human topoisomerase IIα, dovitinib stabilized the enzyme-cleavage complex and acted as a topoisomerase IIα poison. Dovitinib was also found to be a cellular topoisomerase II poison in human leukemia K562 cells and induced double-strand DNA breaks in K562 cells as evidenced by increased phosphorylation of H2AX. Finally, dovitinib inhibited the topoisomerase I-catalyzed relaxation of plasmid DNA and acted as a cellular topoisomerase I poison. In conclusion, the cell growth inhibitory activity and the anticancer activity of dovitinib may result not only from its ability to inhibit multiple kinases, but also, in part, from its ability to target topoisomerase I and topoisomerase II.
Keywords: Dovitinib; Anticancer; DNA; Topoisomerase II; Topoisomerase I
The PPARδ-mediated inhibition of angiotensin II-induced premature senescence in human endothelial cells is SIRT1-dependent by Min Young Kim; Eun Sil Kang; Sun Ah Ham; Jung Seok Hwang; Tae Sik Yoo; Hanna Lee; Kyung Shin Paek; Chankyu Park; Hoon Taek Lee; Jin-Hoi Kim; Chang Woo Han; Han Geuk Seo (pp. 1627-1634).
Cellular senescence has been implicated in endothelial dysfunctions affecting vascular tone and regeneration. The molecular mechanisms of vascular senescence are poorly understood. The present study demonstrates that upregulation of SIRT1 by peroxisome proliferator-activated receptor (PPAR) δ attenuates premature senescence in angiotensin (Ang) II-treated human coronary artery endothelial cells (HCAECs). Activation of PPARδ by the specific ligand GW501516 significantly inhibited Ang II-induced premature senescence and generation of reactive oxygen species (ROS) in HCAECs. A marked concentration- and time-dependent increase in the mRNA levels of SIRT1 was observed in GW501516-treated HCAECs. The effects of GW501516 were almost completely abolished in the presence of small interfering (si) RNA against PPARδ, indicating that PPARδ mediates the effects of GW501516. In addition, activation of PPARδ, but not PPARα or PPARγ, significantly enhanced SIRT1 promoter activity and protein expression. Down-regulation or inhibition of SIRT1 by siRNA or sirtinol abrogated the effects of PPARδ on Ang II-induced premature senescence and ROS generation, respectively. Furthermore, resveratrol, a well-known activator of SIRT1, mimicked the action of PPARδ on Ang II-induced premature senescence and ROS generation. Taken together, these results indicate that the anti-senescent activities of PPARδ may be achieved at least in part by fine tuning the expression of SIRT1 in the vascular endothelium.
Keywords: Abbreviations; Ang II; angiotensin II; HCAECs; human coronary artery endothelial cells; PPAR; peroxisome proliferator-activated receptor; PPRE; PPAR response elements; ROS; reactive oxygen species; SA-β-gal; senescence-associated β-galactosidase; SIPS; stress-induced premature senescence; sir2; silent information regulator 2; TGF; transforming growth factor; VSMCs; vascular smooth muscle cellsAngiotensin II; Peroxisome proliferator-activated receptor δ; Premature senescence; Reactive oxygen species; SIRT1
The PPARδ-mediated inhibition of angiotensin II-induced premature senescence in human endothelial cells is SIRT1-dependent by Min Young Kim; Eun Sil Kang; Sun Ah Ham; Jung Seok Hwang; Tae Sik Yoo; Hanna Lee; Kyung Shin Paek; Chankyu Park; Hoon Taek Lee; Jin-Hoi Kim; Chang Woo Han; Han Geuk Seo (pp. 1627-1634).
Cellular senescence has been implicated in endothelial dysfunctions affecting vascular tone and regeneration. The molecular mechanisms of vascular senescence are poorly understood. The present study demonstrates that upregulation of SIRT1 by peroxisome proliferator-activated receptor (PPAR) δ attenuates premature senescence in angiotensin (Ang) II-treated human coronary artery endothelial cells (HCAECs). Activation of PPARδ by the specific ligand GW501516 significantly inhibited Ang II-induced premature senescence and generation of reactive oxygen species (ROS) in HCAECs. A marked concentration- and time-dependent increase in the mRNA levels of SIRT1 was observed in GW501516-treated HCAECs. The effects of GW501516 were almost completely abolished in the presence of small interfering (si) RNA against PPARδ, indicating that PPARδ mediates the effects of GW501516. In addition, activation of PPARδ, but not PPARα or PPARγ, significantly enhanced SIRT1 promoter activity and protein expression. Down-regulation or inhibition of SIRT1 by siRNA or sirtinol abrogated the effects of PPARδ on Ang II-induced premature senescence and ROS generation, respectively. Furthermore, resveratrol, a well-known activator of SIRT1, mimicked the action of PPARδ on Ang II-induced premature senescence and ROS generation. Taken together, these results indicate that the anti-senescent activities of PPARδ may be achieved at least in part by fine tuning the expression of SIRT1 in the vascular endothelium.
Keywords: Abbreviations; Ang II; angiotensin II; HCAECs; human coronary artery endothelial cells; PPAR; peroxisome proliferator-activated receptor; PPRE; PPAR response elements; ROS; reactive oxygen species; SA-β-gal; senescence-associated β-galactosidase; SIPS; stress-induced premature senescence; sir2; silent information regulator 2; TGF; transforming growth factor; VSMCs; vascular smooth muscle cellsAngiotensin II; Peroxisome proliferator-activated receptor δ; Premature senescence; Reactive oxygen species; SIRT1
Rosuvastatin reduces platelet recruitment by inhibiting NADPH oxidase activation by Pasquale Pignatelli; Roberto Carnevale; Serena Di Santo; Simona Bartimoccia; Cristina Nocella; Tommasa Vicario; Lorenzo Loffredo; Francesco Angelico; Francesco Violi (pp. 1635-1642).
Rosuvastatin increased vascular endothelial NO and attenuated platelet activation after ischemia-reperfusion in mice; nevertheless, the influence of rosuvastatin on the activation of human platelets and the underlying mechanism has never been investigated.In an in vitro study platelets from 8 healthy donors were incubated with scalar concentrations of rosuvastatin (1–10μM) before activation. Platelet recruitment (PR), that mimics the propagation of platelet aggregation and is dependent upon isoprostane formation, was investigated. PR was inhibited by rosuvastatin in concentration-dependent manner concomitantly with down-regulation of platelet release of the pro-thrombotic molecule CD40L. This effect was associated with lower production of platelet reactive oxygen species (ROS), isoprostane and activation of the glycoprotein IIb/IIIa and was counteracted by exogenous addition of isoprostanes. Conversely, rosuvastatin concentration-dependently increased platelet NO. Platelet isoprostane formation mainly depends from NADPH oxidase. Rosuvastatin concentration-dependently inhibited platelet sNOX2-dp release, a specific marker of NADPH oxidase activation, PKC phosphorylation and p47phox translocation from cytosol to membranes.In an ex vivo study 10 hypercolesterolemic patients were randomly allocated to diet or rosuvastatin (20mg). We observed that as early as 2h after rosuvastatin PR, platelet isoprostanes formation, platelet CD40L and sNOX2-dp decreased while platelet NO increased; no changes were detected in diet-assigned patients.This study shows that in vitro rosuvastatin impairs platelet activation via inhibition of NOX2-derived oxidative stress. This effect, which is associated ex vivo with acute inhibition of platelet activation, suggests that rosuvastatin behaves as an antiplatelet drug.
Keywords: Platelets; Oxidative stress; NADPH oxidase; Statins
Rosuvastatin reduces platelet recruitment by inhibiting NADPH oxidase activation by Pasquale Pignatelli; Roberto Carnevale; Serena Di Santo; Simona Bartimoccia; Cristina Nocella; Tommasa Vicario; Lorenzo Loffredo; Francesco Angelico; Francesco Violi (pp. 1635-1642).
Rosuvastatin increased vascular endothelial NO and attenuated platelet activation after ischemia-reperfusion in mice; nevertheless, the influence of rosuvastatin on the activation of human platelets and the underlying mechanism has never been investigated.In an in vitro study platelets from 8 healthy donors were incubated with scalar concentrations of rosuvastatin (1–10μM) before activation. Platelet recruitment (PR), that mimics the propagation of platelet aggregation and is dependent upon isoprostane formation, was investigated. PR was inhibited by rosuvastatin in concentration-dependent manner concomitantly with down-regulation of platelet release of the pro-thrombotic molecule CD40L. This effect was associated with lower production of platelet reactive oxygen species (ROS), isoprostane and activation of the glycoprotein IIb/IIIa and was counteracted by exogenous addition of isoprostanes. Conversely, rosuvastatin concentration-dependently increased platelet NO. Platelet isoprostane formation mainly depends from NADPH oxidase. Rosuvastatin concentration-dependently inhibited platelet sNOX2-dp release, a specific marker of NADPH oxidase activation, PKC phosphorylation and p47phox translocation from cytosol to membranes.In an ex vivo study 10 hypercolesterolemic patients were randomly allocated to diet or rosuvastatin (20mg). We observed that as early as 2h after rosuvastatin PR, platelet isoprostanes formation, platelet CD40L and sNOX2-dp decreased while platelet NO increased; no changes were detected in diet-assigned patients.This study shows that in vitro rosuvastatin impairs platelet activation via inhibition of NOX2-derived oxidative stress. This effect, which is associated ex vivo with acute inhibition of platelet activation, suggests that rosuvastatin behaves as an antiplatelet drug.
Keywords: Platelets; Oxidative stress; NADPH oxidase; Statins
The (pro)renin receptor ((P)RR) can act as a repressor of Wnt signalling by Sarah M. Bernhard; Kerstin Seidel; Jennifer Schmitz; Sabrina Klare; Sebastian Kirsch; Eva Schrezenmeier; Daniela Zaade; Heike Meyborg; Petra Goldin-Lang; Philipp Stawowy; Frank S. Zollmann; Thomas Unger; Heiko Funke-Kaiser (pp. 1643-1650).
The (pro)renin receptor ((P)RR) and Wnt signalling are both involved in different diseases ranging from cardiac and renal end-organ damage to cancer. (P)RR function involves signalling via the transcription factor promyelocytic leukemia zinc finger protein (PLZF) as well as the furin-mediated generation of vacuolar proton-translocating ATPase (V-ATPase)-associated and soluble (P)RR isoforms. Recently, the (P)RR was described as adaptor protein of Wnt (co)receptors. The aim of this study was to analyse the contribution of these distinct (P)RR functions to Wnt signalling.Using Tcf/Lef reporter gene systems in HEK293T and HepG2 cells and quantification of endogenous axin2 mRNA and protein levels in HEK293T cells we were able to demonstrate that full-length (P)RR acts as a repressor of Wnt signalling in a system preactivated either by Wnt3a stimulation or by constitutively active β-catenin. These repressive effects are mediated by Dvl but are independent of the mutation status of β-catenin. Furthermore, the V-ATPase complex, but not PLZF translocation or renin enzymatic activity, is necessary for the induction of Tcf/Lef-responsive genes by Wnt3a.Our data indicate interference of (P)RR and Wnt cascades, a fact that has to be considered concerning pathophysiology of cardio-renal and oncological entities as well as in drug development programs targeting (P)RR or Wnt pathways.
Keywords: (Pro)renin receptor; Wnt; Dvl; Beta-catenin; Furin; Aliskiren
The (pro)renin receptor ((P)RR) can act as a repressor of Wnt signalling by Sarah M. Bernhard; Kerstin Seidel; Jennifer Schmitz; Sabrina Klare; Sebastian Kirsch; Eva Schrezenmeier; Daniela Zaade; Heike Meyborg; Petra Goldin-Lang; Philipp Stawowy; Frank S. Zollmann; Thomas Unger; Heiko Funke-Kaiser (pp. 1643-1650).
The (pro)renin receptor ((P)RR) and Wnt signalling are both involved in different diseases ranging from cardiac and renal end-organ damage to cancer. (P)RR function involves signalling via the transcription factor promyelocytic leukemia zinc finger protein (PLZF) as well as the furin-mediated generation of vacuolar proton-translocating ATPase (V-ATPase)-associated and soluble (P)RR isoforms. Recently, the (P)RR was described as adaptor protein of Wnt (co)receptors. The aim of this study was to analyse the contribution of these distinct (P)RR functions to Wnt signalling.Using Tcf/Lef reporter gene systems in HEK293T and HepG2 cells and quantification of endogenous axin2 mRNA and protein levels in HEK293T cells we were able to demonstrate that full-length (P)RR acts as a repressor of Wnt signalling in a system preactivated either by Wnt3a stimulation or by constitutively active β-catenin. These repressive effects are mediated by Dvl but are independent of the mutation status of β-catenin. Furthermore, the V-ATPase complex, but not PLZF translocation or renin enzymatic activity, is necessary for the induction of Tcf/Lef-responsive genes by Wnt3a.Our data indicate interference of (P)RR and Wnt cascades, a fact that has to be considered concerning pathophysiology of cardio-renal and oncological entities as well as in drug development programs targeting (P)RR or Wnt pathways.
Keywords: (Pro)renin receptor; Wnt; Dvl; Beta-catenin; Furin; Aliskiren
ABCC2 is involved in the hepatocyte perinuclear barrier for small organic compounds by Ruben Rosales; Maria J. Monte; Alba G. Blazquez; Oscar Briz; Jose J.G. Marin (pp. 1651-1659).
Small organic molecules are believed to freely diffuse across nuclear pores but this may not be so if this route is blocked during protein and nucleic acid transfer. Here we have investigated the existence of transport mechanisms across the nuclear envelope (NE) of hepatocytes. Using nuclei isolated from rat liver cells, evidence for the existence of ATP-dependent transporters of organic compounds was found. In rat hepatocyte NE, with negligible contamination by other membranes, the presence of mature and glycosylated ABCC2, but not other ABC export pumps, was detected. ABCC2 was localized in the same membranes as the conjugating enzyme UGT1A1. Human ABCC2 ORF was tagged with V5 and transfected to human hepatoma cells. ABCC2-V5 protein was detected at perinuclear ER vesicles and at the NE. Both compartments expressing ABCC2-V5 were able to exclude calcein. ABCC2 abundance at the NE of rat hepatocytes was modified by treatments able to increase or reduce the expression of canalicular ABCC2. The sensitivity to mitoxantrone was higher for hepatocytes obtained from TR- rats whose NE lacked ABCC2. Incubation with mitoxantrone after depletion of ATP resulted in a marked accumulation of mitoxantrone in the nucleus of wild-type, but not TR-, hepatocytes. In sum, ABCC2 is present at the NE and perinuclear ER where, in combination with the activity of conjugating enzymes, this pump may be involved in the perinuclear barrier for small organic molecules, playing a role in protecting DNA from genotoxic compounds and in the control of intranuclear concentrations of ligands for nuclear receptors.
Keywords: Abbreviations; ABC; ATP-binding cassette; BDL; bile duct ligation; CAT; chloramphenicol acetyltransferase; CDDP; cisplatin; CGamF; cholylglycylamidofluorescein; FITC; fluorescein isothiocyanate; MRP; multidrug resistance-associated protein; MTX; mitoxantrone; NE; nuclear envelope; NPC; nuclear pore complex; UDCGamF; ursodeoxycholylglycylamidofluoresceinABC proteins; Bilirubin; Mitoxantrone; MRP2; Liver toxicity
ABCC2 is involved in the hepatocyte perinuclear barrier for small organic compounds by Ruben Rosales; Maria J. Monte; Alba G. Blazquez; Oscar Briz; Jose J.G. Marin (pp. 1651-1659).
Small organic molecules are believed to freely diffuse across nuclear pores but this may not be so if this route is blocked during protein and nucleic acid transfer. Here we have investigated the existence of transport mechanisms across the nuclear envelope (NE) of hepatocytes. Using nuclei isolated from rat liver cells, evidence for the existence of ATP-dependent transporters of organic compounds was found. In rat hepatocyte NE, with negligible contamination by other membranes, the presence of mature and glycosylated ABCC2, but not other ABC export pumps, was detected. ABCC2 was localized in the same membranes as the conjugating enzyme UGT1A1. Human ABCC2 ORF was tagged with V5 and transfected to human hepatoma cells. ABCC2-V5 protein was detected at perinuclear ER vesicles and at the NE. Both compartments expressing ABCC2-V5 were able to exclude calcein. ABCC2 abundance at the NE of rat hepatocytes was modified by treatments able to increase or reduce the expression of canalicular ABCC2. The sensitivity to mitoxantrone was higher for hepatocytes obtained from TR- rats whose NE lacked ABCC2. Incubation with mitoxantrone after depletion of ATP resulted in a marked accumulation of mitoxantrone in the nucleus of wild-type, but not TR-, hepatocytes. In sum, ABCC2 is present at the NE and perinuclear ER where, in combination with the activity of conjugating enzymes, this pump may be involved in the perinuclear barrier for small organic molecules, playing a role in protecting DNA from genotoxic compounds and in the control of intranuclear concentrations of ligands for nuclear receptors.
Keywords: Abbreviations; ABC; ATP-binding cassette; BDL; bile duct ligation; CAT; chloramphenicol acetyltransferase; CDDP; cisplatin; CGamF; cholylglycylamidofluorescein; FITC; fluorescein isothiocyanate; MRP; multidrug resistance-associated protein; MTX; mitoxantrone; NE; nuclear envelope; NPC; nuclear pore complex; UDCGamF; ursodeoxycholylglycylamidofluoresceinABC proteins; Bilirubin; Mitoxantrone; MRP2; Liver toxicity
Metformin reduces airway inflammation and remodeling via activation of AMP-activated protein kinase by Chan Sun Park; Bo-Ram Bang; Hyouk-Soo Kwon; Keun-Ai Moon; Tae-Bum Kim; Ki-Young Lee; Hee-Bom Moon; You Sook Cho (pp. 1660-1670).
Recent reports have suggested that metformin has anti-inflammatory and anti-tissue remodeling properties. We investigated the potential effect of metformin on airway inflammation and remodeling in asthma. The effect of metformin treatment on airway inflammation and pivotal characteristics of airway remodeling were examined in a murine model of chronic asthma generated by repetitive challenges with ovalbumin and fungal-associated allergenic protease. To investigate the underlying mechanism of metformin, oxidative stress levels and AMP-activated protein kinase (AMPK) activation were assessed. To further elucidate the role of AMPK, we examined the effect of 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) as a specific activator of AMPK and employed AMPKα1-deficient mice as an asthma model. The role of metformin and AMPK in tissue fibrosis was evaluated using a bleomycin-induced acute lung injury model and in vitro experiments with cultured fibroblasts. Metformin suppressed eosinophilic inflammation and significantly reduced peribronchial fibrosis, smooth muscle layer thickness, and mucin secretion. Enhanced AMPK activation and decreased oxidative stress in lungs was found in metformin-treated asthmatic mice. Similar results were observed in the AICAR-treated group. In addition, the enhanced airway inflammation and fibrosis in heterozygous AMPKα1-deficient mice were induced by both allergen and bleomycin challenges. Fibronectin and collagen expression was diminished by metformin through AMPKα1 activation in cultured fibroblasts. Therefore metformin reduced both airway inflammation and remodeling at least partially through the induction of AMPK activation and decreased oxidative stress. These data provide insight into the beneficial role of metformin as a novel therapeutic drug for chronic asthma.
Keywords: Airway remodeling; AMP-activated protein kinase (AMPK); 5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR); Metformin; Oxidative stress
Metformin reduces airway inflammation and remodeling via activation of AMP-activated protein kinase by Chan Sun Park; Bo-Ram Bang; Hyouk-Soo Kwon; Keun-Ai Moon; Tae-Bum Kim; Ki-Young Lee; Hee-Bom Moon; You Sook Cho (pp. 1660-1670).
Recent reports have suggested that metformin has anti-inflammatory and anti-tissue remodeling properties. We investigated the potential effect of metformin on airway inflammation and remodeling in asthma. The effect of metformin treatment on airway inflammation and pivotal characteristics of airway remodeling were examined in a murine model of chronic asthma generated by repetitive challenges with ovalbumin and fungal-associated allergenic protease. To investigate the underlying mechanism of metformin, oxidative stress levels and AMP-activated protein kinase (AMPK) activation were assessed. To further elucidate the role of AMPK, we examined the effect of 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) as a specific activator of AMPK and employed AMPKα1-deficient mice as an asthma model. The role of metformin and AMPK in tissue fibrosis was evaluated using a bleomycin-induced acute lung injury model and in vitro experiments with cultured fibroblasts. Metformin suppressed eosinophilic inflammation and significantly reduced peribronchial fibrosis, smooth muscle layer thickness, and mucin secretion. Enhanced AMPK activation and decreased oxidative stress in lungs was found in metformin-treated asthmatic mice. Similar results were observed in the AICAR-treated group. In addition, the enhanced airway inflammation and fibrosis in heterozygous AMPKα1-deficient mice were induced by both allergen and bleomycin challenges. Fibronectin and collagen expression was diminished by metformin through AMPKα1 activation in cultured fibroblasts. Therefore metformin reduced both airway inflammation and remodeling at least partially through the induction of AMPK activation and decreased oxidative stress. These data provide insight into the beneficial role of metformin as a novel therapeutic drug for chronic asthma.
Keywords: Airway remodeling; AMP-activated protein kinase (AMPK); 5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR); Metformin; Oxidative stress
Mast cell apoptosis induced by siramesine, a sigma-2 receptor agonist by Jane Spirkoski; Fabio R. Melo; Mirjana Grujic; Gabriela Calounova; Anders Lundequist; Sara Wernersson; Gunnar Pejler (pp. 1671-1680).
Mast cells (MCs) are well known for their detrimental effects in the context of allergic disorders. Strategies that limit MC function can therefore have a therapeutic value. Previous studies have shown that siramesine, a sigma-2 receptor agonist originally developed as an anti-depressant, can induce cell death in transformed cells through a mechanism involving lysosomal destabilization. Since MCs are remarkably rich in lysosome-like secretory granules we reasoned that MCs might be sensitive to siramesine. Here we show that murine and human MCs are highly sensitive to siramesine. Cell death was accompanied by secretory granule permeabilization, as shown by reduced acridine orange staining and leakage of granule proteases into the cytosol. Wild type siramesine-treated MCs underwent cell death with typical signs of apoptosis but MCs lacking serglycin, a proteoglycan crucial for promoting the storage of proteases within MC secretory granules, died predominantly by necrosis. A dissection of the underlying mechanism suggested that the necrotic phenotype of serglycin−/− cells was linked to defective Poly(ADP-ribose) polymerase-1 degradation. In vivo, siramesine treatment of mice caused a depletion of the MC populations of the peritoneum and skin. The present study shows for the first time that MCs are highly sensitive to apoptosis induced by siramesine and introduces the possibility of using siramesine as a therapeutic agent for treatment of MC-dependent disease.
Keywords: Abbreviations; MC; mast cell; mMCP; mouse mast cell protease; BMMC; bone marrow-derived mast cell; PCMC; peritoneal cell-derived mast cell; PARP-1; Poly(ADP-ribose) polymerase-1; AO; acridine orange; WT; wild type; AnnV; Annexin V; CPA3; carboxypeptidase A3; Ig; immunoglobulin; RT-PCR; reverse transcription polymerase chain reaction; HMC-1; Human Mast Cell Line-1; LAD2; Laboratory of Allergic Diseases-2Mast cells; Apoptosis; Siramesine; Serglycin; Secretory granules
Mast cell apoptosis induced by siramesine, a sigma-2 receptor agonist by Jane Spirkoski; Fabio R. Melo; Mirjana Grujic; Gabriela Calounova; Anders Lundequist; Sara Wernersson; Gunnar Pejler (pp. 1671-1680).
Mast cells (MCs) are well known for their detrimental effects in the context of allergic disorders. Strategies that limit MC function can therefore have a therapeutic value. Previous studies have shown that siramesine, a sigma-2 receptor agonist originally developed as an anti-depressant, can induce cell death in transformed cells through a mechanism involving lysosomal destabilization. Since MCs are remarkably rich in lysosome-like secretory granules we reasoned that MCs might be sensitive to siramesine. Here we show that murine and human MCs are highly sensitive to siramesine. Cell death was accompanied by secretory granule permeabilization, as shown by reduced acridine orange staining and leakage of granule proteases into the cytosol. Wild type siramesine-treated MCs underwent cell death with typical signs of apoptosis but MCs lacking serglycin, a proteoglycan crucial for promoting the storage of proteases within MC secretory granules, died predominantly by necrosis. A dissection of the underlying mechanism suggested that the necrotic phenotype of serglycin−/− cells was linked to defective Poly(ADP-ribose) polymerase-1 degradation. In vivo, siramesine treatment of mice caused a depletion of the MC populations of the peritoneum and skin. The present study shows for the first time that MCs are highly sensitive to apoptosis induced by siramesine and introduces the possibility of using siramesine as a therapeutic agent for treatment of MC-dependent disease.
Keywords: Abbreviations; MC; mast cell; mMCP; mouse mast cell protease; BMMC; bone marrow-derived mast cell; PCMC; peritoneal cell-derived mast cell; PARP-1; Poly(ADP-ribose) polymerase-1; AO; acridine orange; WT; wild type; AnnV; Annexin V; CPA3; carboxypeptidase A3; Ig; immunoglobulin; RT-PCR; reverse transcription polymerase chain reaction; HMC-1; Human Mast Cell Line-1; LAD2; Laboratory of Allergic Diseases-2Mast cells; Apoptosis; Siramesine; Serglycin; Secretory granules
Beta-d-glucoside protects against advanced glycation end products (AGEs)-mediated diabetic responses by suppressing ERK and inducing PPAR gamma DNA binding by Sidhartha K. Mahali; Sunil K. Manna (pp. 1681-1690).
Accumulation of advanced glycation end products (AGEs), due to excessive amounts of 3- or 4-carbon sugars derived from glucose; cause multiple consequences in diabetic patients and older persons. The transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), is down regulated in the diabetic condition. Drugs targeting PPARγ were developed for diabetes therapy. We found that AGE inhibited PPARγ activity in different cell types induced by PPARγ activators, like troglitazone, rosiglitazone, oleamide, and anandamide. AGE induced translocation of PPARγ from nucleus to cytoplasm, increased on activation of ERK in cells. Antioxidants that inhibit AGE-induced NF-κB activation by preventing ROI generation were unable to protect AGE-mediated decrease in PPARγ activity. Only mangiferin, a β-d-glucoside, prevented AGE-mediated decrease in PPARγ activity and inhibited phosphorylation of ERK and cytoplasmic translocation of PPARγ. Mangiferin interacts with PPARγ and enhanced its DNA binding activity as predicted by in silico and shown by in vitro DNA-binding activity. Overall, the data suggest that (i) mangiferin inhibited AGE-induced ERK activation thereby inhibited PPARγ phosphorylation and cytoplasmic translocation; (ii) mangiferin interacts with PPARγ and enhances its DNA-binding ability. With these dual effects, mangiferin can be a likely candidate for developing therapeutic drug against diabetes.
Keywords: Abbreviations; AGEs; advanced glycation end products; DAPI; 4′-6-diamidino-2-phenylindole; HSA; human serum albumin; IκBα; inhibitory subunit of NF-κB; IKK; IκBα kinase; JNK; c-Jun N-terminal kinase; IκBα-DN; dominant negative of IκBα; MBP; myelin basic protein; MGO; methyl glyoxal; NAC; N-acetylcysteine; NE; nuclear extracts; NF-κB; nuclear transcription factor kappa B; PDTC; pyrrolidine dithiocarbamate; PMA; phorbol myristate acetate; PPAR; peroxisome proliferator-activated receptor; RAGE; receptor for AGE; r.m.s.d.; root mean square deviation; ROI; reactive oxygen intermediatesAGE; NF-κB; ERK; PPAR gamma; Diabetes; Mangiferin
Beta-d-glucoside protects against advanced glycation end products (AGEs)-mediated diabetic responses by suppressing ERK and inducing PPAR gamma DNA binding by Sidhartha K. Mahali; Sunil K. Manna (pp. 1681-1690).
Accumulation of advanced glycation end products (AGEs), due to excessive amounts of 3- or 4-carbon sugars derived from glucose; cause multiple consequences in diabetic patients and older persons. The transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), is down regulated in the diabetic condition. Drugs targeting PPARγ were developed for diabetes therapy. We found that AGE inhibited PPARγ activity in different cell types induced by PPARγ activators, like troglitazone, rosiglitazone, oleamide, and anandamide. AGE induced translocation of PPARγ from nucleus to cytoplasm, increased on activation of ERK in cells. Antioxidants that inhibit AGE-induced NF-κB activation by preventing ROI generation were unable to protect AGE-mediated decrease in PPARγ activity. Only mangiferin, a β-d-glucoside, prevented AGE-mediated decrease in PPARγ activity and inhibited phosphorylation of ERK and cytoplasmic translocation of PPARγ. Mangiferin interacts with PPARγ and enhanced its DNA binding activity as predicted by in silico and shown by in vitro DNA-binding activity. Overall, the data suggest that (i) mangiferin inhibited AGE-induced ERK activation thereby inhibited PPARγ phosphorylation and cytoplasmic translocation; (ii) mangiferin interacts with PPARγ and enhances its DNA-binding ability. With these dual effects, mangiferin can be a likely candidate for developing therapeutic drug against diabetes.
Keywords: Abbreviations; AGEs; advanced glycation end products; DAPI; 4′-6-diamidino-2-phenylindole; HSA; human serum albumin; IκBα; inhibitory subunit of NF-κB; IKK; IκBα kinase; JNK; c-Jun N-terminal kinase; IκBα-DN; dominant negative of IκBα; MBP; myelin basic protein; MGO; methyl glyoxal; NAC; N-acetylcysteine; NE; nuclear extracts; NF-κB; nuclear transcription factor kappa B; PDTC; pyrrolidine dithiocarbamate; PMA; phorbol myristate acetate; PPAR; peroxisome proliferator-activated receptor; RAGE; receptor for AGE; r.m.s.d.; root mean square deviation; ROI; reactive oxygen intermediatesAGE; NF-κB; ERK; PPAR gamma; Diabetes; Mangiferin
Monkey liver cytochrome P450 2C19 is involved in R- and S-warfarin 7-hydroxylation by Yoshio Hosoi; Yasuhiro Uno; Norie Murayama; Hideki Fujino; Mitsunori Shukuya; Kazuhide Iwasaki; Makiko Shimizu; Masahiro Utoh; Hiroshi Yamazaki (pp. 1691-1695).
Cynomolgus monkeys are widely used as primate models in preclinical studies. However, some differences are occasionally seen between monkeys and humans in the activities of cytochrome P450 enzymes. R- and S-warfarin are model substrates for stereoselective oxidation in humans. In this current research, the activities of monkey liver microsomes and 14 recombinantly expressed monkey cytochrome P450 enzymes were analyzed with respect to R- and S-warfarin 6- and 7-hydroxylation. Monkey liver microsomes efficiently mediated both R- and S-warfarin 7-hydroxylation, in contrast to human liver microsomes, which preferentially catalyzed S-warfarin 7-hydroxylation. R-Warfarin 7-hydroxylation activities in monkey liver microsomes were not inhibited by α-naphthoflavone or ketoconazole, and were roughly correlated with P450 2C19 levels and flurbiprofen 4-hydroxylation activities in microsomes from 20 monkey livers. In contrast, S-warfarin 7-hydroxylation activities were not correlated with the four marker drug oxidation activities used. Among the 14 recombinantly expressed monkey P450 enzymes tested, P450 2C19 had the highest activities for R- and S-warfarin 7-hydroxylations. Monkey P450 3A4 and 3A5 slowly mediated R- and S-warfarin 6-hydroxylations. Kinetic analysis revealed that monkey P450 2C19 had high Vmax and low K m values for R-warfarin 7-hydroxylation, comparable to those for monkey liver microsomes. Monkey P450 2C19 also mediated S-warfarin 7-hydroxylation with Vmax and Vmax/ K m values comparable to those for recombinant human P450 2C9. R-warfarin could dock favorably into monkey P450 2C19 modeled. These results collectively suggest high activities for monkey liver P450 2C19 toward R- and S-warfarin 6- and 7-hydroxylation in contrast to the saturation kinetics of human P450 2C9-mediated S-warfarin 7-hydroxylation.
Keywords: Abbreviations; P450; general term for cytochrome P450 (EC 1.14.14.1)Cynomolgus monkey; Human; CYP2C19; CYP2C9; Stereoselectivity
Monkey liver cytochrome P450 2C19 is involved in R- and S-warfarin 7-hydroxylation by Yoshio Hosoi; Yasuhiro Uno; Norie Murayama; Hideki Fujino; Mitsunori Shukuya; Kazuhide Iwasaki; Makiko Shimizu; Masahiro Utoh; Hiroshi Yamazaki (pp. 1691-1695).
Cynomolgus monkeys are widely used as primate models in preclinical studies. However, some differences are occasionally seen between monkeys and humans in the activities of cytochrome P450 enzymes. R- and S-warfarin are model substrates for stereoselective oxidation in humans. In this current research, the activities of monkey liver microsomes and 14 recombinantly expressed monkey cytochrome P450 enzymes were analyzed with respect to R- and S-warfarin 6- and 7-hydroxylation. Monkey liver microsomes efficiently mediated both R- and S-warfarin 7-hydroxylation, in contrast to human liver microsomes, which preferentially catalyzed S-warfarin 7-hydroxylation. R-Warfarin 7-hydroxylation activities in monkey liver microsomes were not inhibited by α-naphthoflavone or ketoconazole, and were roughly correlated with P450 2C19 levels and flurbiprofen 4-hydroxylation activities in microsomes from 20 monkey livers. In contrast, S-warfarin 7-hydroxylation activities were not correlated with the four marker drug oxidation activities used. Among the 14 recombinantly expressed monkey P450 enzymes tested, P450 2C19 had the highest activities for R- and S-warfarin 7-hydroxylations. Monkey P450 3A4 and 3A5 slowly mediated R- and S-warfarin 6-hydroxylations. Kinetic analysis revealed that monkey P450 2C19 had high Vmax and low K m values for R-warfarin 7-hydroxylation, comparable to those for monkey liver microsomes. Monkey P450 2C19 also mediated S-warfarin 7-hydroxylation with Vmax and Vmax/ K m values comparable to those for recombinant human P450 2C9. R-warfarin could dock favorably into monkey P450 2C19 modeled. These results collectively suggest high activities for monkey liver P450 2C19 toward R- and S-warfarin 6- and 7-hydroxylation in contrast to the saturation kinetics of human P450 2C9-mediated S-warfarin 7-hydroxylation.
Keywords: Abbreviations; P450; general term for cytochrome P450 (EC 1.14.14.1)Cynomolgus monkey; Human; CYP2C19; CYP2C9; Stereoselectivity
Rat CYP24A1 acts on 20-hydroxyvitamin D3 producing hydroxylated products with increased biological activity by Elaine W. Tieu; Edith K.Y. Tang; Jianjun Chen; Wei Li; Minh N. Nguyen; Zorica Janjetovic; Andrzej Slominski; Robert C. Tuckey (pp. 1696-1704).
20-Hydroxyvitamin D3 (20(OH)D3), the major product of CYP11A1 action on vitamin D3, is biologically active and is produced in vivo. As well as potentially having important physiological actions, it is of interest as a therapeutic agent due to its lack of calcemic activity. In the current study we have examined the ability of CYP24A1, the enzyme that inactivates 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), to metabolize 20(OH)D3. Rat CYP24A1 was expressed in Escherichia coli, purified by Ni-affinity chromatography and assayed with substrates incorporated into phospholipid vesicles which served as a model of the inner mitochondrial membrane. In this system CYP24A1 metabolized 1,25(OH)2D3 with a catalytic efficiency 1.4-fold higher than that seen for 25-hydroxyvitamin D3 (25(OH)D3). CYP24A1 hydroxylated 20(OH)D3 to several dihydroxy-derivatives with the major two identified by NMR as 20,24-dihydroxyvitamin D3 (20,24(OH)2D3) and 20,25-dihydroxyvitamin D3 (20,25(OH)2D3). The catalytic efficiency of CYP24A1 for 20(OH)D3 metabolism was more than 10-fold lower than for either 25(OH)D3 or 1,25(OH)2D3 and no secondary metabolites were produced. The two major products, 20,24(OH)2D3 and 20,25(OH)2D3, caused significantly greater inhibition of colony formation by SKMEL-188 melanoma cells than either 1,25(OH)2D3 or the parent 20(OH)D3, showing that CYP24A1 plays an activating, rather than an inactivating role on 20(OH)D3.
Keywords: CYP24A1; Vitamin D metabolism; 20-Hydroxyvitamin D; Phospholipid vesicles; MelanomaAbbreviations; (20(OH)D; 3; ); 20; S; -hydroxyvitamin D; 3; (25(OH)D; 3; ); 25-hydroxyvitamin D; 3; (20,24(OH); 2; D; 3; ); 20; S; ,24-dihydroxyvitamin D; 3; (20,25(OH); 2; D; 3; ); 20; S; ,25-dihydroxyvitamin D; 3; (20,26(OH); 2; D; 3; ); 20; S; ,26-dihydroxyvitamin D; 3; (1,25(OH); 2; D; 3; ); 1α,25-dihydroxyvitamin D; 3; ESI; electrospray ionization; HSQC; heteronuclear single quantum correlation; cyclodextrin; 2-hydroxypropyl-β-cyclodextrin
Rat CYP24A1 acts on 20-hydroxyvitamin D3 producing hydroxylated products with increased biological activity by Elaine W. Tieu; Edith K.Y. Tang; Jianjun Chen; Wei Li; Minh N. Nguyen; Zorica Janjetovic; Andrzej Slominski; Robert C. Tuckey (pp. 1696-1704).
20-Hydroxyvitamin D3 (20(OH)D3), the major product of CYP11A1 action on vitamin D3, is biologically active and is produced in vivo. As well as potentially having important physiological actions, it is of interest as a therapeutic agent due to its lack of calcemic activity. In the current study we have examined the ability of CYP24A1, the enzyme that inactivates 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), to metabolize 20(OH)D3. Rat CYP24A1 was expressed in Escherichia coli, purified by Ni-affinity chromatography and assayed with substrates incorporated into phospholipid vesicles which served as a model of the inner mitochondrial membrane. In this system CYP24A1 metabolized 1,25(OH)2D3 with a catalytic efficiency 1.4-fold higher than that seen for 25-hydroxyvitamin D3 (25(OH)D3). CYP24A1 hydroxylated 20(OH)D3 to several dihydroxy-derivatives with the major two identified by NMR as 20,24-dihydroxyvitamin D3 (20,24(OH)2D3) and 20,25-dihydroxyvitamin D3 (20,25(OH)2D3). The catalytic efficiency of CYP24A1 for 20(OH)D3 metabolism was more than 10-fold lower than for either 25(OH)D3 or 1,25(OH)2D3 and no secondary metabolites were produced. The two major products, 20,24(OH)2D3 and 20,25(OH)2D3, caused significantly greater inhibition of colony formation by SKMEL-188 melanoma cells than either 1,25(OH)2D3 or the parent 20(OH)D3, showing that CYP24A1 plays an activating, rather than an inactivating role on 20(OH)D3.
Keywords: CYP24A1; Vitamin D metabolism; 20-Hydroxyvitamin D; Phospholipid vesicles; MelanomaAbbreviations; (20(OH)D; 3; ); 20; S; -hydroxyvitamin D; 3; (25(OH)D; 3; ); 25-hydroxyvitamin D; 3; (20,24(OH); 2; D; 3; ); 20; S; ,24-dihydroxyvitamin D; 3; (20,25(OH); 2; D; 3; ); 20; S; ,25-dihydroxyvitamin D; 3; (20,26(OH); 2; D; 3; ); 20; S; ,26-dihydroxyvitamin D; 3; (1,25(OH); 2; D; 3; ); 1α,25-dihydroxyvitamin D; 3; ESI; electrospray ionization; HSQC; heteronuclear single quantum correlation; cyclodextrin; 2-hydroxypropyl-β-cyclodextrin
Corrigendum to “A novel fibrinogenase from Agkistrodon acutus venom protects against DIC via direct degradation of thrombosis and activation of protein C” [Biochem. Pharmacol. 84 (2012) 905–913]
by Jie-zhen Qi; Xi Lin; Jia-shu Chen; Zhen-hua Huang; Bi-tao Qiu; Peng-xin Qiu; Guang-mei Yan (pp. 1705-1705).
Corrigendum to “A novel fibrinogenase from Agkistrodon acutus venom protects against DIC via direct degradation of thrombosis and activation of protein C” [Biochem. Pharmacol. 84 (2012) 905–913]
by Jie-zhen Qi; Xi Lin; Jia-shu Chen; Zhen-hua Huang; Bi-tao Qiu; Peng-xin Qiu; Guang-mei Yan (pp. 1705-1705).