Effects Of Nucleoside Analogs Research Articles

Effects of nucleoside analogues, lipophilic prodrugs and elaidic acids on core signaling pathways in cancer cells

Objectives Nucleoside analogs such as gemcitabine (GEM; dFdC) and cytarabine (Ara-C) require nucleoside transporters to enter cells, and deficiency in equilibrative nucleoside transporter 1 (ENT1) can lead to resistance to these drugs. To facilitate transport-independent uptake, prodrugs with a fatty acid chain attached to the 5’-position of the ribose group of gemcitabine or cytarabine were developed (CP-4126 and CP-4055, respectively). As antimetabolites can activate cellular survival pathways, we investigated whether the prodrugs or their side-chains had similar or decreased effects. Methods Two cell lines A549 (non-small cell lung cancer) and WiDr (colon cancer cells) were exposed for 2-24hr to IC50 concentrations of GEM, Ara-C, CP-4126, CP4055 and elaidic acid (EA) concentrations corresponding to the CP-4126 and CP-4055 IC50. Cells were harvested and analyzed for proteins in cell survival pathways (p-AKT/AKT, p-ERK/ERK, p-P38/P38, GSK-3β/pGSK-3β) by using Western Blotting. Results All drugs and their derivatives showed time- and cell-line-dependent effects. In A549 cells, GEM, CP-4126 and EA-4126 decreased the p-AKT/AKT ratio at 2 and 24 hr. For the p-ERK/ERK ratio, GEM, EA-4126, Ara-C, CP-4045 and EA-4055 exposure led to an increase after 6 hr in A549 cells. Interestingly, Ara-C, CP-4055 and EA-4055 decreased p-ERK/ERK ratio in WiDr cells after 4 hr. In A549 cells, the p-GSK-3β/GSK-3β ratio decreased after exposure to Ara-C and CP-4055 but in WiDr cells increased after 24 hr. In A549 cells treatment with Ara-C, CP-4055 and EA-4126 decreased the p-P38/P38 after 6 hr. Conclusions The findings suggest that both parent drugs, prodrugs, and the EA chain influence cell survival and signaling pathways.

Nucleos(t)ide analogs for hepatitis B virus infection differentially regulate the growth factor signaling in hepatocytes.

Recent clinical studies have suggested that the risk of developing HCC might be lower in patients with chronic hepatitis B receiving tenofovir disoproxil fumarate than in patients receiving entecavir, although there is no difference in biochemical and virological remission between the 2 drugs. The effects of nucleoside analogs (NsAs; lamivudine and entecavir) or nucleotide analogs (NtAs; adefovir disoproxil, tenofovir disoproxil fumarate, and tenofovir alafenamide) on cell growth and the expression of growth signaling molecules in hepatoma cell lines and PXB cells were investigated in vitro. The tumor inhibitory effects of NsAs or NtAs were evaluated using a mouse xenograft model, and protein phosphorylation profiles were investigated. The binding of NsAs or NtAs to the insulin receptor (INSR) was investigated by thermal shift assays. NtAs, but not NsAs, showed direct growth inhibitory effects on hepatoma cell lines in vitro and a mouse model in vivo. A phosphoprotein array revealed that INSR signaling was impaired and the levels of phosphorylated (p)-INSRβ and downstream molecules phosphorylated (p)-IRS1, p-AKT, p-Gab1, and p-SHP2 were substantially reduced by NtAs. In addition, p-epidermal growth factor receptor and p-AKT levels were substantially reduced by NtAs. Similar findings were also found in PXB cells and nontumor lesions of liver tissues from patients with chronic hepatitis B. Prodrug NtAs, but not their metabolites (adefovir, adefovir monophosphate, adefovir diphosphate, tenofovir, tenofovir monophosphate, and tenofovir diphosphate), had such effects. A thermal shift assay showed the binding of NtAs to INSRβ. NtAs (adefovir disoproxil, tenofovir disoproxil fumarate, and tenofovir alafenamide), which are adenine derivative acyclic nucleotide analogs, potentially bind to the ATP-binding site of growth factor receptors and inhibit their autophosphorylation, which might reduce the risk of HCC in patients with chronic hepatitis B.

Mathematical model of immune response to hepatitis B

A new detailed mathematical model for dynamics of immune response to hepatitis B is proposed, which takes into account contributions from innate and adaptive immune responses, as well as cytokines. Stability analysis of different steady states is performed to identify parameter regions where the model exhibits clearance of infection, maintenance of a chronic infection, or periodic oscillations. Effects of nucleoside analogues and interferon treatments are analysed, and the critical drug efficiency is determined.

Study on 2-arylthio-5-iodo pyrimidine derivatives as novel nonnucleoside inhibitors against hepatitis B virus DNA replication.

Novel nonnucleoside hepatitis B virus inhibitors have been recently developed for the reason of drug-resistant mutations and adverse effects of nucleoside analogs. In this study, two series of 2-arylthio-5-iodo pyrimidine analogs were firstly reported as potential anti-HBV agents. Target compounds were prepared according to two high-yielded synthetic routes, and their anti-HBV activities were evaluated on Hep2.2.15 and HepAD38 cell lines, respectively. To probe the mechanism of active agents, a cell-based (Huh-7) study of biochemical markers (e.g., HBeAg, HBsAg, intracellular HBV DNA and pgRNA) was performed. Furthermore, the pharmacophore models were constructed for future optimization of lead compounds. 2-Arylthio-5-iodo pyrimidine derivatives firstly proved to be effective against HBV, which paves the way for future development of nonnucleoside anti-HBV agents.

Effects of Nucleoside Analogue on Patients with Chronic Hepatitis B-Associated Liver Failure: Meta-Analysis

PurposeThe effectiveness of nucleoside analogue on patients with chronic hepatitis B-associated liver failure is still controversial. To address this issue, we did a review of the literatures and analyzed the data with emphasis on the survival and reduction in serum HBV DNA level.MethodsWe searched 11 randomized controlled trials that included 654 patients with chronic hepatitis B-associated liver failure. 340 patients adopted nucleoside analogue, such as lamivudine (LAM), entecavir (ETV), telbivudine (LdT), or tenofovir disoproxil fumarate (TDF), and the remaining 314 patients adopted no nucleoside analogue or placebo. A meta-analysis was carried out to examine the survival, HBV e antigen serologic conversion, and reduction in serum HBV DNA level. The pooled odds ratio (OR) was used to reflect the treatment effects.ResultsThe overall analysis revealed nucleoside analogue significantly improved 1-month(OR = 2.10; 95% CI, [1.29, 3.41]; p = 0.003), 3-month (OR = 2.15; 95% CI, [1.26, 3.65]; p = 0.005), 12-month survival (OR = 4.62; 95% CI, [1.96, 10.89]; p = 0.0005). Comparison of 3-month HBV DNA showed significant reduction for adoptive nucleoside analogue patients (OR = 54.47; 95% CI, [16.37, 201.74]; p<0.00001). Comparison of 3-month HBV e antigen serologic conversion showed a highly significant improvement of HBV e antigen lost for patients received adoptive antiviral therapy (OR = 6.57; 95% CI, [1.64, 26.31]; p = 0.008).ConclusionsThe benefits of nucleoside analogue on patients with chronic hepatitis B-associated liver failure is significant for improving patient survival, HBV e antigen serologic conversion, and rapid reduction of HBV DNA levels.

Aquaporin 3 (AQP3) participates in the cytotoxic response to nucleoside-derived drugs

BackgroundNucleoside analogs used in the chemotherapy of solid tumors, such as the capecitabine catabolite 5′-deoxy-5-fluorouridine (5′-DFUR) trigger a transcriptomic response that involves the aquaglyceroporin aquaporin 3 along with other p53-dependent genes. Here, we examined whether up-regulation of aquaporin 3 (AQP3) mRNA in cancer cells treated with 5′-DFUR represents a collateral transcriptomic effect of the drug, or conversely, AQP3 participates in the activity of genotoxic agents.MethodsThe role of AQP3 in cell volume increase, cytotoxicity and cell cycle arrest was analyzed using loss-of-function approaches.Results5′-DFUR and gemcitabine, but not cisplatin, stimulated AQP3 expression and cell volume, which was partially and significantly blocked by knockdown of AQP3. Moreover, AQP3 siRNA significantly blocked other effects of nucleoside analogs, including G1/S cell cycle arrest, p21 and FAS up-regulation, and cell growth inhibition. Short incubations with 5-fluorouracil (5-FU) also induced AQP3 expression and increased cell volume, and the inhibition of AQP3 expression significantly blocked growth inhibition triggered by this drug. To further establish whether AQP3 induction is related to cell cycle arrest and apoptosis, cells were exposed to long incubations with escalating doses of 5-FU. AQP3 was highly up-regulated at doses associated with cell cycle arrest, whereas at doses promoting apoptosis induction of AQP3 mRNA expression was reduced.ConclusionsBased on the results, we propose that the aquaglyceroporin AQP3 is required for cytotoxic activity of 5’-DFUR and gemcitabine in the breast cancer cell line MCF7 and the colon adenocarcinoma cell line HT29, and is implicated in cell volume increase and cell cycle arrest.

Erythrocyte Inosine Triphosphatase Activity Is Decreased in HIV-Seropositive Individuals

BackgroundInosine triphosphatase (ITPase) is encoded by the polymorphic gene ITPA and maintains low intracellular levels of the inosine nucleotides ITP and dITP. The most frequently reported polymorphisms are ITPA c.94C>A (rs 1127354) and ITPA c. 124+21 A>C (rs7270101). Some nucleoside-analogues used in the treatment of HIV-seropositive (HIV+) patients are potential substrates for ITPase. Therefore, the frequency of ITPA SNPs and ITPase activity were studied in a population of HIV+-patients.MethodsThe study population consisted of 222 patients, predominantly Caucasian males, >95% using HAART. Erythrocyte ITPase activity was determined by measuring the formation of IMP from ITP. ITPA genotype was determined by sequencing genomic DNA. Distribution of ITPase activity, genotype-phenotype correlation and allele frequencies were compared to 198 control subjects. The effect of nucleoside analogues on ITPase activity was studied using lymphoblastic T-cell cultures and human recombinant ITPase. Enzyme kinetic experiments were performed on erythrocyte ITPase from HIV+ patients and controls.ResultsNo difference was observed in the allele frequencies between the HIV+-cohort (± HAART) and the control population. HIV+ carriers of the wild type and ITPA c.94C>A had significantly lower ITPase activities than control subjects with the same genotype (p<0.005). This was not observed in ITPA c. 124+21 A>C carriers. Nucleoside analogues did not affect ITPase activity in cell culture and human recombinant ITPase. Conclusion: ITPA population genetics were identical in HIV+ and control populations. However, the majority of HIV+-patients had decreased erythrocyte ITPase activity compared to controls, probably due to decreased amounts of ITPase protein. It seems unlikely that ITPase activity is decreased due to nucleoside analogues (HAART). Long-term effects of HIV-infection altering ITPase protein expression or stability may explain the phenomenon observed.

Metabolic limitations of the use of nucleoside analogs in cancer therapy may be overcome by application of nanoparticles as drug carriers: A review

Purine and pyrimidine nucleoside analogs (NAs) are antimetabolites commonly used in cancer therapy. Administered as prodrugs, NAs permeate the mambrane using specialized transporters. Following phosphorylation, they interfere with multiple cellular pocessess inducing cytotoxicity. Toxic effects of NAs are dependent on metabolic conversion from a prodrug into the active form. For instance, an exceptionally high activity of deoxycytidine kinase (dCK) in lymphocytes is correlated with a good therapeutic effect of fludarabine in leukemic cells. On the other hand, several studies have shown that nucleoside-transporter (NT)-deficient-cells are highly resistant to NAs. Attempts to overcome the metabolic limitations of chemotherapeutics would result in the use of lower drug doses for effective therapy, reduce side effects, and ensure the independence of drug resistance mechanisms. To meet this need, several nanoparticles have been designed to deliver efficently NAs directly to cancer cells. Such vehicles include liposomes, albumins, and dendritic and linear polymers. Therapeutic agents encapsulated or conjugated to nanoparticles have improved pharmacokinetics, solubility, and stability. These factors improve the efficacy of commonly used drugs. Moreover, modification of nanoparticles with targeting molecules such as sugar moieties or folic acid ensures more specific delivery without affecting healthy tissues. Drug Dev Res 71: 383–394, 2010. © 2010 Wiley-Liss, Inc.

Human polymerase α inhibitors for skin tumors. Part 2. Modeling, synthesis and influence on normal and transformed keratinocytes of new thymidine and purine derivatives

Recently, the three-dimensional structure of the active site of human DNA polymerase α (pol α) was proposed based on the application of molecular modeling methods and molecular dynamic simulations. The modeled structure of the enzyme was used for docking selective inhibitors (nucleotide analogs and the non-nucleoside inhibitor aphidicolin) in its active site in order to design new drugs for actinic keratosis and squamous cell carcinoma (SCC). The resulting complexes explained the geometrical and physicochemical interactions of the inhibitors with the amino acid residues involved in binding to the catalytic site, and offered insight into the experimentally derived binding data. The proposed structures were synthesized and tested in vitro for their influence on human keratinocytes and relevant tumor cell lines. Effects were compared to aphidicolin which inhibits pol α in a non-competitive manner, as well as to diclofenac and 5-fluorouracil, both approved for therapy of actinic keratosis. Here we describe three new nucleoside analogs inhibiting keratinocyte proliferation by inhibiting DNA synthesis and inducing apoptosis and necrosis. Thus, the combination of modeling studies and in vitro tests should allow the derivation of new drug candidates for the therapy of skin tumors, given that the agents are not relevant substrates of nucleotide transporters expressed by skin cancer cells. Kinases for nucleoside activation were detected, too, corresponding with the observed effects of nucleoside analogs.

The Effects of Nucleoside Analogues on Promoter Methylation of Selected Tumor Suppressor Genes in MCF-7 and MDA-MB-231 Breast Cancer Cell Lines

The effects of 2-chloro-2′-deoxyadenosine, 9-β-D-arabinofuranosyl-2-fluoroadenine, and 5-aza-2′-deoxycytidine on promoter methylation of the selected tumor suppressor genes (i.e., ERα, BRCA1, RARβ2, E-cadherin, PTEN, and APC) were estimated using methylation-sensitive restriction analysis. The studies were carried out in hormone-responsive, low-invasive cell line MCF-7 and hormone-insensitive, highly invasive cell line MDA-MB-231. The results demonstrate an implication of the tested adenosine analogues and 5-aza-dCyd in regulation of DNA methylation process. Moreover, the effects of nucleoside analogues on PTEN promoter methylation suggest distinct mechanism of regulation of the epigenetic DNA modification in low-invasive compared to highly invasive breast cancer cells.

Influence of nucleoside analogue treatment on telomere length and TRF2 amount in human HL60 cells

Long-term treatment with 3'-azido-2',3'-dideoxy-guanosine (AZddG) results in reproducible telomere shortening in cultured human HL60 cells. TRF2 protein has been implicated in the protection of chromosome ends. It binds to double-strand repeats and may have an indirect role in protecting the G-rich overhang by recruiting other telomere-binding proteins to the G-tail or by mediating the formation of the telomeric t-loop. Western blot analysis demonstrated no change or a slight increase, of the TRF2 protein level in HL60 cells with AZddG-induced telomere shortening. The effects of nucleoside analogues on TRF2 suggest that it is not telomere length per se, but rather the presence or absence of a protective telomere state, which determines whether senescence ensues.

Effects of Nucleoside Analogues on Liver Regeneration 70% Partially Hepatectomized Rats

The alternatives for prophlaxis and treatment of recurrent hepatitis B virus infection have increased since new oral nucleoside analogues have become available. We conducted this experimental study to investigate the effect in the liver of these agents on the expression of transforming growth factor alpha (TGF-α) and on proliferation index, estimated by Ki-67. Thirty male Wistar albino rats were randomized into three groups: group A ( n = 10) received adefovir dipivoxil (40 mg/kg/d per gavage); group B ( n = 10), lamivudine (L; 30 mg/kg/d per gavage); and group C ( n = 10) did not receive any treatment and were the control group. Groups A and B were treated for 3 days. Animal treatment began on day −1. After performing 70% partial hepatectomy on day 0, all rats were sacrificed on postoperative day 2 to harvest liver tissues for histopathological examination. We stained and indexed Ki-67 and TGF-α immunohistochemically on the hepatectomy surface and in the parenchyma, Ki-67 and TGF-α indices were significantly higher in group A compared with group B ( P = .001 and P = .004, respectively, and P = .003 and P = .001, respectively). When the L group was compared with the control group for results on the hepatectomy surface and the parenchyma, Ki-67 and TGF-α indexes were insignificantly different ( P = .6 and P = .3, respectively, and P = .1 and P = .6, respectively). Based on the results of this experimental study, we concluded that Adefovir dipivoxil has greater proliferative effect on liver parenchyma and in the cut surface than does lamivudine.

A prospective study on chemotherapy-induced hepatitis B virus reactivation in chronic HBs Ag carriers with hematologic malignancies and pre-emptive therapy with nucleoside analogues

Chemotherapy-induced hepatitis B virus (HBV) reactivation is a serious problem in chronic HBV carriers with hematologic malignancies. In 12 patients with hematologic malignancies, we performed a prospective study to determine the effectiveness of nucleoside analogues in the pre-emptive therapy of chemotherapy-induced HBV reactivation. HBV reactivation occurred in seven patients (58.3%) whereas five of the seven patients (71%) responded to nucleoside analogue therapy. HBV reactivation-related acute liver failure and death was not observed in the present study. All five patients with chronic lymphocytic leukemia (CLL) experienced chemotherapy-induced HBV reactivation regardless of the chemotherapy regimen. Therefore, we suggest that CLL carries a significant risk of chemotherapy-induced HBV reactivation. The pre-emptive therapy of chemotherapy-induced HBV reactivation appears to be safe, based on the results of this pilot study. Pre-emptive therapy enables the definition of high-risk patients who cannot be identified by primary prophylaxis.

Old and new insights into the mechanisms of action of two nucleoside analogs active in lymphoid malignancies: fludarabine and cladribine (Review)

This review focuses on two chemotherapeutic agents belonging to the family of purine analogs, 9-beta-D-arabinosyl-2-fluoroadenine-5'-monophosphate (F-ara-AMP, fludarabine), which is the soluble form of F-ara-A, and 2-chloro-2'-deoxyadenosine (CdA, cladribine). Both compounds display remarkable activity in malignancies arising from the clonal expansion of lymphocytes, and particularly in B-cell chronic lymphocytic leukemia (B-CLL). These analogs are prodrugs that must be converted into their respective nucleotides as an essential requirement for cytotoxicity. Triphosphate analogs inhibit various processes involved in DNA and RNA synthesis. Moreover, the effect of nucleoside analogs in leukemic cells may involve modulation of apoptosis pathways, cell-cycle control, or signal transduction pathways. These findings are of crucial importance because alterations in these pathways may be involved in leukemic cell resistance toward nucleoside analogs and, hence, constitute new molecular targets to sensitize leukemic cells to these drugs.

A role for MAP kinase in the antitumor activity of a nucleoside analog.

Nucleoside analogs (NAs) have been used extensively in both antitumor and antiviral therapies. Their general mechanism of action has been postulated to result from incorporation into DNA, leading to disruption of DNA synthesis and DNA polymerase inhibition. To further explore the antitumor mechanisms of NAs we have evaluated ganciclovir (GCV), an NA antiviral agent, in herpes simplex virus thymidine kinase (HSV-TK) gene-modified tumor cells. This system allows specific evaluation of the antitumor effects of NAs because the antitumor effect is directly related to the phosphorylation of the prodrug GCV by the HSV-TK enzyme in the gene-modified tumor cells. We demonstrated that GCV incorporates into DNA and inhibits DNA polymerase, as has been observed in HSV-infected cells and with other antitumor NAs in tumor cells. A novel observation is that GCV activates MAP kinase within 1 hour of GCV exposure. This activation directly correlates with cytotoxicity, because inhibition of the MAP kinase extracellular regulated kinase (Erk) by PD98059, reversed GCV-mediated cytotoxicity. This effect appears to be specific to the Erk pathway, because inhibition of the p38 kinase with SB203580 had no effect on cytotoxicity. Further, GCV does not act as a DNA-damaging agent or activate general DNA-repair mechanisms, but does produce a number of metabolic disruptions, including a reversible decrease in NAD levels. These effects appear to be downstream of the earlier activation of Erk in this system, which may be a novel mechanism of action for GCV cytotoxicity in HSV-TK gene-modified tumor cells, and thus, needs to be further evaluated as the mechanism of tumor cell killing by other antitumor NAs.

Effects of Nucleoside Analogs on Native and Site-Directed Mutants of HTLV Type 1 Reverse Transcriptase

A bacterial assay was developed for testing HTLV-1 reverse transcriptase sensitivity to common nucleoside analog inhibitors in an Escherichia coli strain characterized by a temperature sensitive PolI/RecA deletion phenotype. This genetic complementation assay exploits the ability of HTLV-1 reverse transcriptase to functionally replace these missing activities at nonpermissive temperatures. The four inhibitors tested, dideoxyinosine, dideoxyadenosine, deoxythymidine, and didehydrodeoxythymidine are well-known inhibitors of HIV reverse transcriptase. All except dideoxyadenosine showed a strong activity against HTLV-1 reverse transcriptase with IC50; in the nanomolar range. Sequence alignments were used to identify amino acid residues in HTLV-1 reverse transcriptase, which correspond to those identified as important for drug-resistance in HIV reverse transcriptase. Mutations of some of these HTLV-1 residues altered the IC50 for the inhibitors as expected, which suggests that these amino acids have a function in HTLV-1 reverse transcriptase similar to that of their homologs in HIV reverse transcriptase.

Effect of nucleoside analogues and oligonucleotides on hydrolysis of liposomal phospholipids

The hydrolysis of the bilayer forming phospholipids resulting first of all in lysophospholipids and fatty acids is one limiting factor determining the shelf-life of liposomes. In several studies the influence of pH, buffer, lipid composition and other parameters on the hydrolysis of phospholipids have been demonstrated, but the influence of drugs has not yet been investigated systematically. In this study the influence of nucleoside analogues, especially 2′,2′-difluoro 2′-deoxycytidine (gemcitabine, dFdC) on the degradation of phospholipids was elucidated in more detail. It could be demonstrated that the interaction of dFdC with phospholipid bilayers promotes the hydrolysis of phospholipids in a concentration-dependent manner. Obviously two parts of the molecule, the amino group bound to the pyrimidine moiety and the 2′,2′-difluoro-2′-deoxyribose, seem to be responsible for the forced phospholipid hydrolysis. The dFdC-induced hydrolysis of phospholipids was influenced by pH, buffer, lipid composition and different anions. Optimization of the above parameters resulted in prolonged shelf-life of dFdC liposome dispersions, which is an important prerequisite for clinical practice.

Effect of nucleoside analogs and non-nucleoside inhibitors of HIV-1 reverse transcriptase on cell-free virions.

Reverse transcription takes place in the cytoplasm of infected cells, although it has been demonstrated that retroviruses can also initiate reverse transcription prior to infection of target cells. In addition to partial reverse transcripts, full-length proviral molecules have been detected in the plasma and seminal fluid of HIV-1 seropositive patients. Intravirion endogenous reverse transcription appears to be directly correlated with an increased level of infectivity. Therefore, the ability of an inhibitor to reach and inhibit the replication complex in the core of the free-virion may constitute an important part of its capacity to suppress viral infection. In this work we tested the ability of some reverse transcriptase inhibitors to decrease viral infectivity in pretreated highly purified virions. Our results showed that Curie pyridinone [Dollé et al. (1995), J Med Chem 38: 4,679-4,686], a non nucleoside RT inhibitor, strongly inhibited the infectivity of extracellular HIV-1 particles. Other non nucleoside inhibitors (TIBO R82913, HEPT, nevirapine) tested in these conditions were unable to do so. Our data indicate that the effect of Curie pyridinone on intact virions may be related to its capacity to tightly bind the target RT. This approach may lead to the design and synthesis of new drugs able to interact with the retroviral enzyme inside the viral core.

The Use of 31P Relaxation Experiments to Probe the Effects of Nucleoside Analogs on DNA Dynamics

The Use of 31P Relaxation Experiments to Probe the Effects of Nucleoside Analogs on DNA Dynamics

The Use of 31P Relaxation Experiments to Probe the Effects of Nucleoside Analogs on DNA Dynamics

In the present study, we examine the effects of two nucleoside analogs on the structure and dynamics of self-complementary duplexes and model primer/template duplexes.