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1.
Hepatology ; 58(4): 1236-44, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23532970

ABSTRACT

UNLABELLED: Ribavirin (RBV) is often used in conjunction with interferon-based therapy for patients with chronic hepatitis C. There is a drastic difference in the anti-hepatitis C virus (HCV) activity of RBV between the HuH-7-derived assay system, OR6, possessing the RBV-resistant phenotype (50% effective concentration [EC50 ]: >100 µM) and the recently discovered Li23-derived assay system, ORL8, possessing the RBV-sensitive phenotype (EC50 : 8 µM; clinically achievable concentration). This is because the anti-HCV activity of RBV was mediated by the inhibition of inosine monophosphate dehydrogenase in RBV-sensitive ORL8 cells harboring HCV RNA. By means of comparative analyses using RBV-resistant OR6 cells and RBV-sensitive ORL8 cells, we tried to identify host factor(s) determining the anti-HCV activity of RBV. We found that the expression of adenosine kinase (ADK) in ORL8 cells was significantly higher than that in RBV-resistant OR6 cells harboring HCV RNA. Ectopic ADK expression in OR6 cells converted them from an RBV-resistant to an RBV-sensitive phenotype, and inhibition of ADK abolished the activity of RBV. We showed that the differential ADK expression between ORL8 and OR6 cells was not the result of genetic polymorphisms in the ADK gene promoter region and was not mediated by a microRNA control mechanism. We found that the 5' untranslated region (UTR) of ADK messenger RNA in ORL8 cells was longer than that in OR6 cells, and that only a long 5' UTR possessed internal ribosome entry site (IRES) activity. Finally, we demonstrated that the long 5' UTR functioned as an IRES in primary human hepatocytes. CONCLUSION: These results indicate that ADK acts as a determinant for the activity of RBV and provide new insight into the molecular mechanism underlying differential drug sensitivity.


Subject(s)
Adenosine Kinase/physiology , Antiviral Agents/pharmacology , Hepacivirus/drug effects , Hepatitis C/pathology , Hepatocytes/drug effects , Ribavirin/pharmacology , Antiviral Agents/therapeutic use , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/virology , Cell Line, Tumor , Drug Resistance, Viral , Hepacivirus/genetics , Hepatitis C/drug therapy , Hepatitis C/metabolism , Hepatocytes/pathology , Hepatocytes/virology , Humans , Liver Neoplasms/pathology , Liver Neoplasms/virology , Phenotype , RNA, Viral/metabolism , Ribavirin/therapeutic use , Treatment Outcome
2.
J Proteome Res ; 11(12): 5704-11, 2012 Dec 07.
Article in English | MEDLINE | ID: mdl-23061985

ABSTRACT

The endoperoxide artemisinin is a current first-line antimalarial and a critical component of the artemisinin-based combination therapies (ACT) recommended by WHO for treatment of Plasmodium falciparum, the deadliest of malaria parasites. However, recent emergence of the artemisinin-resistant P. falciparum urged us to develop new antimalarial drugs. We have shown that synthetic endoperoxides N-89 and its hydroxyl derivative N-251 had high antimalarial activities both in vivo and in vitro. However, the mechanisms including the cellular targets of the endoperoxide antimalarials are not well understood. Thus, in this study, we employed chemical proteomics to survey potential molecular targets of endoperoxides by evaluating P. falciparum proteins capable to associate with endoperoxide structure (N-346, a carboxyamino derivative of N-89). We also analyzed the protein expression profiles of malaria parasites treated with N-89 or N-251 to explore possible changes associated with the drug action. From these experiments, we found that P. falciparum endoplasmic reticulum-resident calcium binding protein (PfERC) had high affinity to the endoperoxide structure (N-346) and was decreased by treatment with N-89 or N-251. PfERC is a member of CREC protein family, a potential disease marker and also a potential target for therapeutic intervention. We propose that the PfERC is a strong candidate of the endoperoxide antimalarial's target.


Subject(s)
Antimalarials/pharmacology , Calcium-Binding Proteins/chemistry , Endoplasmic Reticulum/chemistry , Peroxides/pharmacology , Plasmodium falciparum/chemistry , Protozoan Proteins/chemistry , Antimalarials/chemistry , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Erythrocytes/parasitology , Heterocyclic Compounds, 2-Ring/pharmacology , Humans , Peroxides/chemistry , Plasmodium falciparum/drug effects , Plasmodium falciparum/pathogenicity , Proteomics/methods , Recombinant Proteins/chemistry , Spiro Compounds/pharmacology , Tetraoxanes/pharmacology , Trophozoites/chemistry , Trophozoites/drug effects
3.
Parasitol Int ; 60(4): 488-92, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21924377

ABSTRACT

Malaria is one of the world's deadliest diseases and is becoming an increasingly serious problem as malaria parasites develop resistance to most of the antimalarial drugs used today. We previously reported the in vitro and in vivo antimalarial potencies of 1,2,6,7-tetraoxaspiro[7.11]nonadecane (N-89) and 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251) against Plasmodium falciparum and Plasmodium berghei parasites. To improve water-solubility for synthetic peroxides, a variety of cyclic peroxides having carboxyl functionality was prepared based on the antimalarial candidate, N-251, and their antimalarial activities were determined. The reactions of N-89 and its derivatives with Fe(II) demonstrated a highly efficient formation of the corresponding carbon radical which may be suspected as a key for the antiparasitic activity.


Subject(s)
Antimalarials/administration & dosage , Hexanols/administration & dosage , Malaria, Falciparum/drug therapy , Malaria/drug therapy , Plasmodium berghei/drug effects , Plasmodium falciparum/drug effects , Spiro Compounds/administration & dosage , Animals , Antimalarials/chemical synthesis , Antimalarials/therapeutic use , Carbon/chemistry , Carbon/metabolism , Carboxylic Acids/chemistry , Drug Evaluation, Preclinical , Ferrous Compounds/metabolism , Free Radicals/chemistry , Free Radicals/metabolism , Hexanols/chemical synthesis , Hexanols/therapeutic use , Humans , Inhibitory Concentration 50 , Malaria/parasitology , Malaria, Falciparum/parasitology , Mice , Mice, Inbred ICR , Oxidation-Reduction , Peroxides/chemistry , Peroxides/metabolism , Plasmodium berghei/growth & development , Plasmodium falciparum/growth & development , Spiro Compounds/chemical synthesis , Spiro Compounds/therapeutic use , Structure-Activity Relationship
4.
Parasitol Int ; 60(3): 270-3, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21501696

ABSTRACT

Plasmodium falciparum, the major causative parasite for the disease, has acquired resistance to most of the antimalarial drugs used today, presenting an immediate need for new antimalarial drugs. Here, we report the in vitro and in vivo antimalarial activities of 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251) against P. falciparum and Plasmodium berghei parasites. The N-251 showed high antimalarial potencies both in the in vitro and the in vivo tests (EC(50) 2.3×10(-8) M; ED(50) 15 mg/kg (per oral)). The potencies were similar to that of artemisinin in vitro and greater than artemisinin's activity in vivo (p.o.). In addition, N-251 has little toxicity: a single oral administration at 2000 mg/kg to a rat gave no health problems to it. Administration of N-251 to mice bearing 1% of parasitemia (per oral 68 mg/kg, 3 times a day for 3 consecutive days) resulted in a dramatic decrease in the parasitemia: all the 5 mice given N-251 were cured without any recurrence, with no diarrhea or weight loss occurring in the 60 days of experiment. N-251 deserves more extensive clinical evaluation, desirably including future trials in the human.


Subject(s)
Antimalarials/pharmacology , Artemisinins/pharmacology , Heterocyclic Compounds, 2-Ring/pharmacology , Hexanols/pharmacology , Malaria, Falciparum/drug therapy , Plasmodium berghei/drug effects , Plasmodium falciparum/drug effects , Spiro Compounds/pharmacology , Administration, Oral , Animals , Antimalarials/chemistry , Antimalarials/therapeutic use , Artemisinins/therapeutic use , Cell Line, Tumor , Drug Therapy, Combination , Erythrocytes/drug effects , Erythrocytes/parasitology , Heterocyclic Compounds, 2-Ring/chemical synthesis , Heterocyclic Compounds, 2-Ring/therapeutic use , Hexanols/chemistry , Hexanols/therapeutic use , Humans , Malaria/drug therapy , Malaria/parasitology , Malaria, Falciparum/parasitology , Mice , Mice, Inbred ICR , Molecular Structure , Parasitemia/drug therapy , Parasitic Sensitivity Tests , Rats , Spiro Compounds/chemical synthesis , Spiro Compounds/chemistry , Spiro Compounds/therapeutic use , Survival Analysis , Tetraoxanes
5.
Nucleic Acids Symp Ser (Oxf) ; (52): 627-8, 2008.
Article in English | MEDLINE | ID: mdl-18776536

ABSTRACT

We report that anticancer 5-fluoro-2'-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A cells, using a progeny clone F28-7 and its variant F28-7-A. In this process, the cell-death morphology is different between F28-7 and F28-7-A cells, that is, necrosis in F28-7 but apoptosis in F28-7-A cells. Recently we have investigated the gene and protein expression profiles of necrosis and apoptosis induced by FUdR using transcriptomic and proteomic analysis. In the proteomic analysis of these cells before their exposure to FUdR, the nuclear inner-membrane protein lamin B1 is up-regulated in F28-7 but not in F28-7-A, suggesting that lamin B1 may possess a function to regulate the morphology of cell-death. A knockdown of lamin B1 expression in F28-7 cells has now been performed by use of the small interfering RNA technique, resulting in a decrease of the lamin B1-expression level down to the level in F28-7-A. Remarkably, the FUdR-induced death morphology of this knocked-down F28-7 was apoptosis, definitely different from the necrosis that occurs in the FudR-treated original F28-7. This finding suggests a new role for lamin B1 as a regulator in the cell death.


Subject(s)
Antimetabolites, Antineoplastic/toxicity , Apoptosis/physiology , Floxuridine/toxicity , Necrosis/metabolism , Animals , Cell Line, Tumor , Lamin Type B/antagonists & inhibitors , Lamin Type B/genetics , Lamin Type B/metabolism , Mice , Necrosis/genetics , RNA Interference
6.
Genomics ; 92(1): 9-17, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18572099

ABSTRACT

5-Fluoro-2'-deoxyuridine (FUdR), a potent anticancer agent, exerts its effects by inhibiting thymidylate synthase, an essential machinery for DNA synthesis in cell proliferation. Also, cell death is caused by FUdR, primarily due to an imbalance in the nucleotide pool resulting from this enzyme inhibition. We have investigated the cancer cell death induced by FUdR, focusing on its molecular mechanisms. Using mouse mammary tumor FM3A cell lines, the original clone F28-7 and its variant F28-7-A cells, we previously reported an interesting observation that FUdR induces a necrotic morphology in F28-7, but induces, in contrast, an apoptotic morphology in F28-7-A cells. In the present study, to understand the molecular mechanisms underlying these differential cell deaths, i.e., necrosis and apoptosis, we investigated the gene expression changes occurring in these processes. Using the cDNA microarray technology, we found 215 genes being expressed differentially in the necrosis and apoptosis. Further analysis revealed differences between these cell lines in terms of the expressions of both a cluster of heat shock protein (HSP)-related genes and a cluster of apoptosis-related genes. Notably, inhibition of HSP90 in F28-7 cells caused a shift from the FUdR-induced necrosis into apoptosis. These findings are expected to lead to a better understanding of this anticancer drug FUdR for its molecular mechanisms and also of the general biological issue, necrosis and apoptosis.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Apoptosis/genetics , Floxuridine/pharmacology , Gene Expression Profiling , Necrosis/chemically induced , Necrosis/genetics , Animals , Cell Line, Tumor , Cytochromes c/metabolism , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Mice , Mitochondria/drug effects , Mitochondria/enzymology , Necrosis/metabolism , Oligonucleotide Array Sequence Analysis
7.
Nucleic Acids Symp Ser (Oxf) ; (51): 433-4, 2007.
Article in English | MEDLINE | ID: mdl-18029772

ABSTRACT

5-fluoro-2'-deoxyuridine (FUdR) inhibits thymidylate synthase. We have been investigated the molecular mechanisms of cell death in mouse mammary tumor FM3A cells, F28-7 strain and its mutant F28-7-A strain, after treated with FUdR. Previously, we have been reported that F28-7 strain induced DNA cleavage into chromosomal sized fragments and subsequently develop necrosis, but F28-7-A strain induced DNA cleavage into oligonucleosomal sized fragments and subsequently develop apoptosis after treated with FUdR. To understand the molecular mechanisms of regulate of two differential cell death necrosis and apoptosis, we identify cell death regulator by using proteome and transcriptome analysis. When compared with the proteome of F28-7 and F28-7-A strain after treated with FUdR, it was found that 5 proteins were up-regulated and 11 proteins were down-regulated in F28-7-A strain. Furthermore, transcriptome analysis shows that 94 genes were up-regulated and 164 genes were downregulated in F28-7-A strain. Identified proteins and genes were involved in various cellular processes such as cell cycle regulation, apoptosis, proliferation, and differentiation. Our results suggested that numerous features indicated the coordinated regulation of molecular networks from various aspects of necrosis or apoptosis at the proteome and transcriptome levels.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Apoptosis , Floxuridine/pharmacology , Animals , Apoptosis/genetics , Cell Line, Tumor , Gene Expression Profiling , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/metabolism , Mammary Neoplasms, Experimental/pathology , Mice , Necrosis , Proteomics
8.
Parasitol Res ; 100(5): 1119-24, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17273878

ABSTRACT

N-89, a new antimalarial endoperoxide, was selected as a promising antimalarial compound showing high activity and selectivity. To study the mechanism of N-89 action, N-89 resistant strain (NRC10) was obtained by intermittent drug pressure. NRC10 had a tenfold increase in the EC(50) value of N-89. No cross-resistance was obtained with other antimalarial compounds. Comparative proteome analysis of N-89 sensitive and NRC10 strains revealed over-expression of 12 spots and down-regulation of 14 spots in NRC10. Fifteen proteins were identified of Plasmodium falciparum origin. The identified proteins representing several functions, mainly related to the glycolytic pathway, and metabolism of protein and lipid. Our results suggest that identified proteins may be candidates of antimalarial endoperoxide targets.


Subject(s)
Antimalarials/pharmacology , Peroxides/pharmacology , Plasmodium falciparum/chemistry , Plasmodium falciparum/drug effects , Proteome/drug effects , Protozoan Proteins/analysis , Animals , Drug Resistance , Electrophoresis, Gel, Two-Dimensional , Parasitic Sensitivity Tests
9.
Nucleic Acids Symp Ser (Oxf) ; (50): 101-2, 2006.
Article in English | MEDLINE | ID: mdl-17150837

ABSTRACT

5-Fluoro-2'-deoxyuridine (FUdR) inhibits thymidylate synthase. The inhibition of thymidylate synthase causes an imbalance of intracellular deoxyribonucleoside triphosphate (dNTP) pools which subsequently induced cell death. We have been investigated the molecular mechanisms of cell death in mouse mammary tumor FM3A cells, F28-7 strain and its mutant F28-7-A strain, after treated with FUdR. We have previously been reported that F28-7 strain induced DNA cleavage into chromosomal sized fragments and subsequently develop necrosis, but F28-7-A strain induced DNA cleavage into oligonucleosomal sized fragments and subsequently develop apoptosis after treated with FUdR. In this report, in order to understand the molecular mechanisms of regulate of two differential cell death necrosis and apoptosis, we identify cell death regulator by using proteome and transcriptome analysis. When compared with the proteome from F28-7 strain and F28-7-A strain, it was found that ten proteins were increased and six proteins were decreased in F28-7-A strain. Furthermore, transcriptome analysis shows that 127 genes were increased and 181 genes were decreased in F28-7-A strain. These differentially expressed proteins and genes were involved in various cellular processes such as cell cycle regulation, apoptosis, proliferation, and differentiation. These two techniques clarified numerous features in F28-7 strain and F28-7-A strain. Our results revealed that numerous features indicated the coordinated regulation of molecular networks from various aspects of necrosis or apoptosis at the proteome and transcriptome levels.


Subject(s)
Antimetabolites, Antineoplastic/toxicity , Apoptosis , Floxuridine/toxicity , Necrosis , Proteome/metabolism , Transcription, Genetic/drug effects , Animals , Cell Line, Tumor , Gene Expression Profiling , Mice , Proteomics
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