Anticancer Strategy Targeting Cell Death Regulators: Switching the Mechanism of Anticancer Floxuridine-Induced Cell Death from Necrosis to Apoptosis.

Cell death can be broadly characterized as either necrosis or apoptosis, depending on the morphological and biochemical features of the cell itself. We have previously reported that the treatment of mouse mammary carcinoma FM3A cells with the anticancer drug floxuridine (FUdR) induces necrosis in the original clone F28-7 but apoptosis in the variant F28-7-A. We have identified regulators, including heat shock protein 90, lamin-B1, cytokeratin-19, and activating transcription factor 3, of cell death mechanisms by using comprehensive gene and protein expression analyses and a phenotype-screening approach. We also observed that the individual inhibition or knockdown of the identified regulators in F28-7 results in a shift from necrotic to apoptotic morphology. Furthermore, we investigated microRNA (miRNA, miR) expression profiles in sister cell strains F28-7 and F28-7-A using miRNA microarray analyses. We found that several unique miRNAs, miR-351-5p and miR-743a-3p, were expressed at higher levels in F28-7-A than in F28-7. Higher expression of these miRNAs in F28-7 induced by transfecting miR mimics resulted in a switch in the mode of cell death from necrosis to apoptosis. Our findings suggest that the identified cell death regulators may play key roles in the decision of cell death mechanism: necrosis or apoptosis.

Role of the uridine/cytidine kinase 2 mutation in cellular sensitiveness toward 3'-ethynylcytidine treatment of human cancer cells.

A nucleosidic medicine, 1-(3-C-ethynyl-ß-D-ribo-pentofuranosyl)cytosine [3'-ethynylcytidine (ECyd)], is a potent inhibitor of RNA polymerase I and shows anticancer activity to various human solid tumors in vitro and in vivo. ECyd is phosphorylated to 3'-ethyntlcytidine 5'-monophosphate by uridine/cytidine kinase 2 (UCK2) and subsequently further to diphosphate and triphosphate (3'-ethyntlcytidine 5'-diphosphate, 3'-ethyntlcytidine 5'-triphosphate). 3'-Ethyntlcytidine 5'-triphosphate is an active metabolite that can inhibit RNA polymerase I competitively, causing cancer cell death. Here, to identify the UCK2 mutation for detecting responder or nonresponder to ECyd, we investigated the relationship between point mutation of the UCK2 gene and response to ECyd in various human solid tumors. We identified several functional point mutations including the splice-site mutation of the UCK2 gene IVS5+5 G>A. In addition, we found that the IVS5+5 G>A variant generates an aberrant mRNA transcript, namely, truncated mRNA was produced and normal mRNA levels were markedly decreased in the ECyd-resistant cancer cell line HT1080. We concluded that these findings strongly suggest that the IVS5+5 G>A variant would affect the expression level of the UCK2 transcript, resulting in decreased sensitivity to ECyd.

Improvement of Oral Bioavailability of N-251, a Novel Antimalarial Drug, by Increasing Lymphatic Transport with Long-Chain Fatty Acid-Based Self-Nanoemulsifying Drug Delivery System.

PURPOSE: The objective of this study was to improve the absorption behavior of N-251, a novel antimalarial drug, by preparing an appropriate self-nanoemulsifying drug delivery system (SNEDDS). METHODS: Two different types of SNEDDS formulations, medium-chain fatty acid-based SNEDDS (MC-SNEDDS) and long-chain fatty acid-based SNEDDS (LC-SNEDDS), were prepared based on pseudo-ternary phase diagram, and examined for their in vivo oral absorption behavior in rats. RESULTS: Oral dosing of MC-SNEDDS formulations significantly improved the bioavailability (BA) of N-251 compared with N-251 powders. However, its high hepatic extraction limited the BA of N-251 to only 0.49 for MC-SNEDDS B, the best formulation of MC-SNEDDS. LC-SNEDDS formulations, especially LC-SNEDDS F provided the highest BA, 0.65, and successfully attenuated the inter-individual difference in the absorption behavior. Furthermore, it was confirmed that lymphatic transport of N-251 for LC-SNEDDS F was significantly increased up to around 3.19 times larger than that for MC-SNEDDS B. Simulation study suggested that 20 to 39% of N-251 uptaken by the small intestine would be delivered to lymphatic system after oral administration of LC-SNEDDS F. CONCLUSIONS: SNEDDS formulations significantly improved the absorption behavior of N-251 and long-chain fatty acid-based lipid further improved it by avoiding the hepatic first-pass elimination.

Adenosine kinase is a key determinant for the anti-HCV activity of ribavirin.

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.

Extracellular Leakage Protein Patterns in Two Types of Cancer Cell Death: Necrosis and Apoptosis.

Dead cells release fragments of DNA, RNA, and proteins (including peptides) into the extracellular space. Two major forms of cell death during cancer development have been identified: necrosis and apoptosis. Our group investigated the mechanisms that regulate cell death during the treatment of mouse tumor FM3A cells with the anticancer drug floxuridine (FUdR). In the original strain F28-7, FUdR induced necrosis, whereas in the variant F28-7-A, it induced apoptosis. Here, we report that the extracellular leakage proteome (i.e., the secretome) is involved in these cell death phenomena. The secretome profile, which was analyzed via shotgun proteomic analysis, revealed that altered protein leakage was involved in signal transduction, transcription, RNA processing, translation, and cell death. Notably, the characteristic secretory proteins high mobility group box 1 and 2 were detected in the culture medium of both necrotic and apoptotic cells. Overall, these results indicate that unique cellular events mediated by secretory proteins may be involved in necrosis and apoptosis.

Plasmodium falciparum endoplasmic reticulum-resident calcium binding protein is a possible target of synthetic antimalarial endoperoxides, N-89 and N-251.

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.

Protein expression profiles of necrosis and apoptosis induced by 5-fluoro-2'-deoxyuridine in mouse cancer cells.

We have investigated the molecular mechanisms regulating the necrosis and apoptosis that occur on treatment of mouse mammary tumor FM3A cells with 5-fluoro-2'-deoxyuridine (FUdR), a potent anticancer agent, using the original clone F28-7 and its variant F28-7-A cells. Previously, we reported an interesting observation that FUdR induces a necrotic morphology in F28-7 but an apoptotic morphology in F28-7-A cells. We have now analyzed the protein expression profiles of these FUdR-induced necrosis and apoptosis. Thus, proteome analysis of these clones by two-dimensional gel electrophoresis and mass spectrometry showed that the cytoplasmic intermediate filament protein, cytokeratin-19, is expressed at a significantly higher level in F28-7 than in F28-7-A cells. This strong expression was detected both in untreated and FUdR-treated stages of F28-7 cells. We interpreted this phenomenon as suggesting that cytokeratin-19 possesses a function in leading the cell to apoptosis. We performed a knockdown of cytokeratin-19 expression in F28-7 cells by use of the small interfering RNA technique. Indeed, a lowering of the cytokeratin-19 expression down to the level in F28-7-A occurred, and the FUdR-induced death morphology of this knockdown F28-7 was apoptosis, instead of the necrosis usually observable in the FUdR-treated F28-7. It is known that the cytoskeletal protein cytokeratin-19 undergoes caspase-mediated degradation during apoptosis. Our present finding provides an interesting possibility that cytokeratin-19 may have a key role in regulating cell-death morphology.

Direct interaction analysis of microRNA-351-5p and nuclear scaffold lamin B1 mRNA by the cell-free in vitro mRNA/miRNA binding evaluation system.

We previously demonstrated that miR-351-5p regulates nuclear scaffold lamin B1 expression and mediates the anticancer floxuridine-induced necrosis shift to apoptosis in mammalian tumor cells. Notably, it is unknown whether lamin B1 mRNA is a direct target of miR-351-5p. Here, we show that miR-351-5p interacts with a lamin B1 mRNA partial sequence by using the cell-free in vitro miRNA and mRNA binding evaluation system. In addition, the interaction of miR-351-5p/lamin B1 mRNA was suppressed by an miR-351-5p inhibitor. Our findings are important in exploring the functions of miRNAs in cellular processes, including cell death.

Intracellular microRNA expression patterns influence cell death fates for both necrosis and apoptosis.

MicroRNAs (miRNAs) are small noncoding RNA molecules that interact with target mRNAs at specific sites to induce cleavage of the mRNA or inhibit translation. Such miRNAs play a vital role in gene expression and in several other biological processes, including cell death. We have studied the mechanisms regulating cell death (necrosis in original F28-7 cells and apoptosis in their variant F28-7-A cells) in the mouse mammary tumor cell line FM3A using the anticancer agent floxuridine (FUdR). We previously reported that inhibition of heat-shock protein 90 by the specific inhibitor geldanamycin (GA) in F28-7 cells causes a shift from necrosis to apoptosis. In this study, we investigated the intracellular miRNA expression profiles of FUdR-treated F28-7 cells (necrotic condition), GA plus FUdR-treated F28-7 cells (apoptotic condition), and FUdR-treated F28-7-A cells (apoptotic condition) through miRNA microarray analysis. In addition, we knocked down Dicer, a key molecule for the expression of mature miRNAs, in F28-7 cells to examine whether it modulates FUdR-induced cell death. Our analysis revealed that the miRNA expression patterns differ significantly between these cell death conditions. Furthermore, we identified miRNA candidates that regulate cell death. Knockdown of Dicer in FUdR-treated necrosis-fated cells caused a partial shift from necrosis to apoptosis. These findings suggest that modulation of miRNA expression patterns influences the decision of cell death fate toward necrosis or apoptosis. Our findings may serve as a basis for further study of the functions of miRNAs in cell death mechanisms.

1-(3-C-Ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106), a novel potent inhibitor of RNA polymerase, potentiates the cytotoxicity of CDDP in human cancer cells both in vitro and in vivo.

1-(3-C-Ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106) is a novel antitumor ribonucleoside that inhibits RNA polymerase. In the present study, we investigated the cellular and molecular interactions between TAS-106 and cisplatin (CDDP) in vitro using A549 human lung cancer cells and the in vivo antitumor effect of combined treatment using OCC-1 and LX-1 human tumor xenografts. The treatment effects were determined by evaluating cytotoxicity, the cell cycle distribution, apoptosis induction and the expression of checkpoint-associated proteins. In vitro, the combination of TAS-106 and CDDP synergistically inhibited the growth of A549 cells, as determined using isobologram analysis. TAS-106 potently inhibited the expression of Chk1 protein and the phosphorylation of Chk1 and Chk2. Moreover, based on the inhibition of checkpoint-associated protein, TAS-106 abrogated the CDDP-induced S- and G2M-checkpoints and induced apoptosis in A549 cells. In vivo, TAS-106 alone showed antitumor activity; however, its combination with CDDP significantly enhanced the growth inhibition of OCC-1 and LX-1 tumors. Moreover, combination therapy with TAS-106 and CDDP in the OCC-1 xenograft model resulted in significant life-prolongation. These findings provide a rationale for combination chemotherapy using TAS-106 and CDDP in clinical settings.

Synthesis of novel conjugates of tetraoxane endoperoxide with bis(quaternary ammonium salts).

Novel water-soluble conjugates of 1,2,4,5-tetraoxane bis(quaternary ammonium salts) were synthesized in a relatively stable crystalline form via four steps starting from methyltrioxorhenium-catalyzed endo-peroxidation of ethyl 4-oxocyclohexanecarboxylate with hydrogen peroxide in hexafluoro-2-propanol. The assay for the in vitro toxicity of water-soluble tetraoxanes 5a-5d to malaria parasites indicate that they were inactive against the Plasmodium falciparum FCR-3 strain.

Toxoplasma gondii: a simple high-throughput assay for drug screening in vitro.

Toxoplasma gondii is the etiologic agent of toxoplasmosis. Although the combination of sulfadiazine and pyrimethamine is used as therapy for this disease, these drugs can have serious side effects and its use is limited in pregnancy. Therefore there is a need for new anti-T. gondii drugs in the clinic. Some systems for T. gondii drug screening have been described, but these have limitations and can be difficult. In order to solve these problems, we established a system to screen drugs in vitro that involved using cell viability methods to calculate drug selectivities, which are Trypan blue, [3-(4,5-dimethylthiazol-zyl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliuzolium, inner salt] (MTS) method and lactate dehydrogenase (LDH) assay. These assays were simple to establish and perform. The IC(50) values calculated from the morphological assay were not significantly different from the EC(50) values calculated using the other three methods. In particular, the results of the morphological assay showed a distinct association with the MTS assay (R=0.9841). These assays could be used for a wide range of applications in the screening of new drugs and may provide an alternative to the techniques currently used to screen for candidate anti-T. gondii compounds in vitro. In this study, we also tested many compounds and identified some that had a good anti-T. gondii effect in vitro based on the MTS assay. This simple and fast system allowed us to determine which compounds to investigate further using in vivo experiments.

Gene expression profiles of necrosis and apoptosis induced by 5-fluoro-2'-deoxyuridine.

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.

Genomic and biological features of Plasmodium falciparum resistance against antimalarial endoperoxide N-89.

Drug resistance of malaria parasites remains a problem affecting antimalarial treatment and control of the disease. We previously synthesized an antimalarial endoperoxide, N-89, having high antimalarial effects in vitro and in vivo. In this study we seek to understand the resistant mechanism against N-89 by establishing a highly N-89-resistant clone, named NRC10H, of the Plasmodium falciparum FCR-3 strain. We describe gene mutations in the parent FCR-3 strain and the NRC10H clone using whole-genome sequencing and subsequently by expression profiling using quantitative real-time PCR. Seven genes related to drug resistance, proteolysis, glycophosphatidylinositol anchor biosynthesis, and phosphatidylethanolamine biosynthesis exhibited a single amino acid substitution in the NRC10H clone. Among these seven genes, the multidrug resistance protein 2 (mdr2) variant A532S was found only in NRC10H. The genetic status of the P. falciparum endoplasmic reticulum-resident calcium binding protein (PfERC), a potential target of N-89, was similar between the NRC10H clone and the parent FCR-3 strain. These findings suggest that the genetic alterations of the identified seven genes, in particular mdr2, in NRC10H could give rise to resistance of the antimalarial endoperoxide N-89.

Synthesis and silencing properties of siRNAs possessing lipophilic groups at their 3'-termini.

Short-interfering RNAs (siRNAs) conjugated with lipophilic groups at their 3'-termini were synthesized. The properties of the synthesized siRNAs were examined in detail, and it was found that at low concentrations, their silencing abilities were dependent on the positions of the modifications and the types of organic molecules attached. Although the modification of siRNAs with palmitic acid or oleic acid at the 3'-end slightly reduced their silencing activities, siRNAs had enough abilities to induce RNAi at 10 nM concentrations. On the other hand, the modification of siRNAs with cholesterol at the 3'-end of the passenger strand was tolerated; however, the modification at the guide strand significantly reduces its silencing activity. The siRNAs modified with the lipophilic groups did not possess ability to penetrate the plasma membranes of HT-1080 cells without the transfection reagent. However, the results described in this report will aid in designing novel siRNAs with cell membrane-permeable molecules.

Association of nuclear membrane protein lamin B1 with necrosis and apoptosis in cell death induced by 5-fluoro-2'-deoxyuridine.

We report that anticancer 5-fluoro-2 '-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A, 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. 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 was 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. Thus, the swelling feature for the necrosis was no longer observable, and instead cell shrinkage typical of apoptosis took place in almost all the cells examined. This finding suggests a new role for lamin B1 as a regulator in cell death.

Potent Antimalarial Activity of Two Arenes Linked with Triamine Designed To Have Multiple Interactions with Heme.

Based on the idea that compounds designed to exhibit high affinity for heme would block hemozoin formation, a critical heme-detoxification process for malarial parasites, we synthesized a series of compounds with two π-conjugated moieties at terminal amino groups of triamine. These compounds exhibited moderate to high antimalarial activities in vitro toward both chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum. In a P. berghei-infected mouse model, 3a and 12a showed potent antimalarial activities compared to artesunate, as well as a prolonged duration of antimalarial effect. We found a good correlation between protective activity against hemin degradation and antimalarial activity. Compounds 8b and 3a strongly inhibited hemozoin formation catalyzed by heme detoxification protein.

Evaluation of preclinical antimalarial drugs, which can overcome direct-acting antivirals-resistant hepatitis C viruses, using the viral reporter assay systems.

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a major global health problem. Recently developed treatments with direct-acting antivirals (DAAs) have largely improved the sustained virologic response rate of patients with chronic hepatitis C. However, this approach is still hindered by its great expense and the problem of drug resistance. Using our cell-based HCV assay systems, we reported that the preclinical antimalarial drugs N-89 and N-251 exhibited potent anti-HCV activities. In this study we used our assay systems to evaluate the anti-HCV activities of six kinds of DAAs individually or in combination with N-89 or N-251. The results showed that the DAAs had potent anti-HCV activities and N-89 or N-251 contributed additive or synergistic effect. Using DAA-resistant HCV-RNA-replicating cells, which were prepared by continuous treatment with each DAA, we demonstrated that N-89 and N-251 could overcome all of the DAA-resistant HCVs. These preclinical drugs would have been potential as components of a therapeutic regimen that also included combinations of various DAAs. In addition, sequence analysis of the NS3-NS5B regions of the DAA-resistant HCV genomes newly found several amino acid (aa) substitutions that were suggested to contribute to DAA-resistance in addition to the aa substitutions already known to cause DAA-resistance. Among these new aa substitutions, we found that two substitutions in the NS3 region (D79G and S174Y) contributed to simeprevir- and/or asunaprevir-resistance.

MicroRNA-351 Regulates Two-Types of Cell Death, Necrosis and Apoptosis, Induced by 5-fluoro-2'-deoxyuridine.

Cell-death can be necrosis and apoptosis. We are investigating the mechanisms regulating the cell death that occurs on treatment of mouse cancer cell-line FM3A with antitumor 5-fluoro-2'-deoxyuridine (FUdR): necrosis occurs for the original clone F28-7, and apoptosis for its variant F28-7-A. Here we report that a microRNA (miR-351) regulates the cell death pattern. The miR-351 is expressed strongly in F28-7-A but only weakly in F28-7. Induction of a higher expression of miR-351 in F28-7 by transfecting an miRNA mimic into F28-7 resulted in a change of the death mode; necrosis to apoptosis. Furthermore, transfection of an miR-351 inhibitor into F28-7-A resulted in the morphology change, apoptosis to necrosis, in this death-by-FUdR. Possible mechanism involving lamin B1 in this miR-351's regulatory action is discussed.

Clinical characteristics of imported malaria in Japan: analysis at a referral hospital.

Imported malaria remains an important problem in Japan. We have reviewed the medical records of 170 cases of malaria in our hospital, which corresponds to 14.9% of the total cases in Japan. The predominant malarial species was Plasmodium falciparum (52.3%), and the most frequent area of acquisition was Africa (54.2%), followed by Asia (20.9%) and Oceania (19.6%). The most common reason for travel among Japanese patients was business. A significant proportion (22.2%) of vivax malaria cases experienced relapse despite standard primaquine therapy. Most primaquine failures were from Oceania. We also found that a substantial number of Japanese patients contracted malaria without chemoprophylaxis and consulted medical facilities with an unfavorably long delay from initial symptoms (median: 3.0 days). Direct education of travelers and travel companies, in addition to health care providers, is likely necessary to improve outcomes of imported malaria.