Identification of chemoresistant factors by protein expression analysis with iTRAQ for head and neck carcinoma.

BACKGROUND: Cisplatin and other anticancer drugs are important in the treatment of head and neck squamous cell carcinoma; however, some tumours develop drug resistance. If chemoresistance could be determined before treatment, unnecessary drug administration would be avoided. Here, we investigated chemoresistance factors by comprehensive analyses at the protein level. METHODS: Four human carcinoma cell lines were used: cisplatin-sensitive UM-SCC-23, UM-SCC-23-CDDPR with acquired cisplatin resistance, naturally cisplatin-resistant UM-SCC-81B, and UM-SCC-23/WR with acquired 5-fluorouracil resistance. Extracted proteins were labelled with iTRAQ and analysed by tandem mass spectrometry to identify resistance. Protein expression was confirmed by western blotting and functional analysis was carried out using siRNA. RESULTS: Thirteen multiple-drug resistance proteins were identified, as well as seven proteins with specific resistance to cisplatin, including α-enolase. Differential expression of these proteins in cisplatin-resistant and -sensitive cell lines was confirmed by western blotting. Functional analysis for α-enolase by siRNA showed that cisplatin sensitivity significantly was increased in UM-SCC-81B and slightly in UM-SCC-23-CDDPR but not in UM-SCC-23/WR cells. CONCLUSIONS: We identified proteins thought to mediate anticancer drug resistance using recent proteome technology and identified α-enolase as a true cisplatin chemoresistance factor. Such proteins could be used as biomarkers for anticancer agent resistance and as targets of cancer therapy.

Acquisition of EMT phenotype in the gefitinib-resistant cells of a head and neck squamous cell carcinoma cell line through Akt/GSK-3ß/snail signalling pathway.

BACKGROUND: Epithelial mesenchymal transition (EMT) is known to be associated with chemoresistance as well as increased invasion/metastasis. However, the relationship between EMT and resistance to an epidermal growth factor receptor (EGFR) -targeting drug in head and neck squamous cell carcinoma (HNSCC) remains unknown. In this study, we investigated the acquisition of EMT by gefitinib in HNSCC cell line (UMSCC81B). METHODS: We isolated fibroblastoid variant (81B-Fb) from gefitinib-resistant UMSCC81B-GR3 cells obtained after increasing the doses of gefitinib treatment in vitro and examined EMT and its underlying mechanism. RESULT: 81B-Fb cells exhibited fibroblast-like morphology, increased motility, loss of E-cadherin, acquisition of vimentin and snail expression. In 81B-Fb cells, downregulation of EGFR, which is mediated by increased ubiquitination, and activation of downstream protein kinase B (Akt), glycogen synthase kinase-beta (GSK-3ß) signalling and upregulation of snail expression were observed compared with UMSCC81B cells. LY294002, but not U0126, suppressed foetal bovine serum or heregulin-ß1-induced phosphorylation of Akt/GSK-3ß and snail expression together with the inhibition of 81B-Fb cell motility. Furthermore, forced expression of EGFR resulted in partial restoration of gefitinib sensitivity and reversal of EMT. CONCLUSION: These results suggest that EMT in the gefitinib-resistant cells is mediated by the downregulation of EGFR and compensatory activation of Akt/GSK-3ß/snail pathway.

[The effects of milrinone on hemodynamics in patients undergoing cardiac surgery].

The phosphodiesterase inhibitor, milrinone is used to treat low cardiac output syndrome, especially after cardiac surgery. But there were few reports about the precise hemodynamic effects at separation from cardiopulmonary bypass (CPB). We examined the hemodynamic effects of milrinone in 24 patients undergoing elective coronary artery bypass graft (CABG). Patients were assigned to the milrinone group (n = 12) and the control group (n = 12). Before separation from CPB, milrinone was administered as a loading dose of 50 micrograms.kg-1 into the reservoir of CPB at rectal temperature 33.5 degrees C and simultaneously a continuous infusion of 0.5 microgram.kg-1.min-1 was started. In addition, dopamine and nitroglycerine were administered in both groups. Hemodynamic measurements were performed before CPB, just after the weaning from CPB, 15, 30, 60 minutes after the weaning from CPB. Cardiac index increased significantly (P < 0.01) in the milrinone group as compared with the control group. Systemic vascular resistance index and mean arterial pressure decreased significantly (P < 0.0001, P < 0.05, respectively) in the milrinone group as compared with the control group. There were no significant differences in heart rate, mean pulmonary arterial pressure, pulmonary artery occlusion pressure, mean right atrial pressure, stroke volume index, and pulmonary vascular resistance index between the two groups. These hemodynamic effects showed that milrinone supported cardiac performance after CPB for CABG.

Inhibition of tumor cell growth by a specific 6-phosphofructo-2-kinase inhibitor, N-bromoacetylethanolamine phosphate, and its analogues.

The high rate of glycolysis despite the presence of oxygen and mitochondria in tumor cells implies an important role for this process in cell division. The rate of glycolysis is assumed to be dependent on the cellular concentration of fructose 2,6-bisphosphate, the concentration of which in turn depends on a bifunctional enzyme and the ratio of this enzyme's 6-phosphofructo-2-kinase versus its fructose 2,6-bisphosphatase activities. To prove the hypothesis that inhibition of glycolysis in tumor cells by 6-phosphofructo-2-kinase inhibitors would cause inhibition of tumor cell proliferation, ten N-bromoacetylethanolamine phosphate analogues were designed, synthesized, and tested. They were screened for their activities against various human tumor cell lines to study the effects of inhibition of glycolysis on cell proliferation. The relationship between the structure of these compounds and their inhibitory activity on cell proliferation was also discussed. It was found that the activity of N-(2-methoxyethyl)-bromoacetamide, N-(2-ethoxyethyl)-bromoacetamide, and N-(3-methoxypropyl)-bromoacetamide was comparable to that of the positive control AraC. These three inhibitors showed in vivo anticancer effects in P388 transplant BDF1 mice.

Upregulation of HIV-1 replication in chronically infected cells by ingenol derivatives.

We have previously reported that ingenol derivatives are highly potent inhibitors of human immunodeficiency virus type 1 (HIV-1) replication in acutely infected cells. In this study, however, we have found that some ingenol derivatives strongly enhance the replication of HIV-1 in chronically infected cells at nanomolar concentrations. One of the derivatives could activate nuclear factor kappa B (NF-kappa B), a potent inducer of HIV-1 replication, through the activation of protein kinase C (PKC). Whereas another derivative, which affected neither PKC nor NF-kappa B, significantly enhanced HIV-1 replication, suggesting that a PKC-independent mechanism may also exist in ingenol derivative-induced HIV-1 upregulation.

Synthesis and antiviral activities of 5-substituted 1-(2-deoxy-2-C-methylene-4-thio-beta-D-ertythro-pentofuranosyl)uracils.

Various 5-substituted 1-(2-deoxy-2-C-methylene-4-thio-beta-D-erythro-pentofuranosyl)uracils (4'-thioDMDUs) were synthesized from D-glucose via sila-Pummerer-type glycosylation. All of the beta-anomers of 5-substituted 4'-thioDMDU, except the 5-hydroxyethyl derivative, showed potent anti-HSV-1 activity (ED50 = 0.016-0.096 microgram/mL). 5-Ethyl- and 5-iodo-4'-thioDMDUs were also active against HSV-2 (ED50 = 0.17 and 0.86 microgram/mL, respectively). 5-Bromovinyl-4'-thioDMDU was particularly active against VZV (ED50 = 0.013 microgram/mL).

Antioxidative properties of products from amino acids or peptides in the reaction with glucose.

The livers of rats fed with a brownish "soybean paste" (Miso) or peptides-glucose reaction mixture showed lower TBA and chemiluminescence values than those of the control. From these results, it was clear that Miso and peptide-glucose reaction products also exhibited antioxidative effect in vivo. In order to explain this mechanism, the scavenging activity against reactive oxygen species (ROS) of various Maillard reaction products (MRPs) were studied. It was confirmed that peptide-glucose reaction products. Amadori rearrangement products, melanoidins, modified protein and its hydrolysate, brown pigments isolated from Miso and other foodstuffs showed strong scavenging activity against hydroxyl radical and superoxide anion. Consequently, it was estimated that scavenging activity of MRPs against ROS played an important role in the antioxidative effect of MRPs in vivo.

A facile, alternative synthesis of 4'-thioarabinonucleosides and their biological activities.

4'-Thioarabinonucleosides, which are potential antiviral agents, were synthesized from D-glucose. 1,4-Anhydro-4-thioarabitol (8), which can be derived from diacetone glucose in nine steps, was subjected to Pummerer rearrangement after protection of the hydroxyl groups to give 1-O-acetyl-4-thioarabinose (11), which was condensed with nucleobases to give 4'-thioarabinonucleosides. The 5-substituted-4'-thioaraU (6a-e) derivatives showed anti-HSV-1 activity (ED50 = 0.43-3.50 micrograms/mL). 4'-ThioaraG (6h) and 2,6-diaminopurine 4'-thioarabinonucleoside (4'-thioaraDAP, 6g) showed antiviral activity against several herpes viruses and were particularly potent against human cytomegalovirus (0.010 and 0.022 microgram/mL, respectively).

Thiadiazole derivatives: highly potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) replications in vitro.

We have recently reported that thiadiazole (TDA) derivatives are highly potent inhibitors of human immunodeficiency virus type 1 (HIV-1) replication. These compounds belong to the family of nonnucleoside reverse transcriptase inhibitors (NNRTIs). In an attempt to develop more effective and pharmacologically favorable compounds, novel TDA derivatives have been synthesized and examined for their anti-HIV-1 activity in vitro. Among them, RD4-2217 was found to be the most potent inhibitor of HIV-1 replication. It inhibited replication of the HTLV-IIIB strain in MT-4 cells at a concentration of 6 nM. RD4-2217 was also inhibitory to clinical isolates and zidovudine-resistant mutants of HIV-1. The combination of RD4-2217 with zidovudine or the protease inhibitor A-75925 synergistically inhibited HIV-1 replication. Studies on the emergence of drug-resistant mutants revealed that, although much higher concentrations (1-10 microM) were required, RD4-2217 completely suppressed the breakthrough of HIV-1 in the supernatants during long-term culturing of infected cells. Furthermore, RD4-2217 at low concentrations (10 or 100 nM), in combination with zidovudine, also completely inhibited viral breakthrough. In addition, RD4-2217 had lower lipophilicity and improved protein binding as compared to its congener RD4-2024 and loviride. These results suggest that RD4-2217, one of the TDA derivatives, is worth pursuing as a candidate drug for the treatment of HIV-1 infections.

Anti-HIV-1 activity of thiadiazole derivatives: structure-activity relationship, reverse transcriptase inhibition, and lipophilicity.

The structure-activity relationship of the non-nucleoside HIV-1-specific reverse transcriptase (RT) inhibitors 4-phenyl-1,2,5-thiadiazol-3-yl N,N-dialkylcarbamate (TDA) derivatives was investigated with respect to their anti-HIV-1 activity, RT inhibition, and lipophilicity. 4-Phenyl-1,2,5-thiadiazol-3-yl N,N-dimethylcarbamate inhibited HIV-1-induced cytopathic effect (CPE) by 50% at a concentration of 28.8 microM in MT-4 cells. The activity increased more than 100-fold when the hydrogens at the 2-position and the 6-position in phenyl moiety were substituted by chlorines. However, the derivative with a chlorine at the 4-position of phenyl moiety did not show any inhibition of HIV-1 replication at its non-toxic concentrations. All of the 4-(2,6-dichlorophenyl)-1,2,5-thiadiazol-3-yl N-methyl-N-alkylcarbamates proved inhibitory to HIV-1 replication in the nanomolar concentration range. The TDA derivatives that showed anti-HIV-1 activity also inhibited RT activity in an enzymatic assay. However, the TDA derivatives did not show any specific inhibition of a non-nucleoside RT inhibitor (NNRTI)-resistant mutant and its RT activity. When the TDA derivatives were examined for their inhibitory effect on HIV-1 replication in the presence of 50% human serum, the activity significantly decreased depending on-their lipophilicity.

Antiviral activities of nucleotide heterodimers against human immunodeficiency virus type 1 in vitro.

Nucleotide heterodimers were synthesized and examined for their inhibitory effects on the replication of human immunodeficiency virus type 1 (HIV-1), including HIV-1 reverse transcriptase (RT) inhibitor-resistant mutants. 3'-Azido-3'-deoxythymidilyl-(5')-phospho-(5')-6-[(3', 5'-dimethylphenyl)thio]-5-ethyl-1-[(2-hydroxyethoxy)methyl]uracil (AZT-P-E-HEPU-dM) and 3'-azido-3'-deoxythymidilyl-(5')-phospho-(5')-2', 3'-dideoxyinosine (AZT-P-ddI) proved to be highly potent and selective inhibitors of HIV-1 (IIIB strain) in MT-4 cells. The mechanism of inhibition by these heterodimers may be attributed to their degradation and the formation of each constituent. AZT-P-E-HEPU-dM was also markedly inhibitory to an AZT-resistant mutant (HIV-1-IIIB/AZT) and an E-HEPU-dM-resistant mutant (HIV-1-IIIB-R). However, AZT-P-ddI was found to have a less inhibitory effect on HIV-1-IIIB/AZT than on HIV-1-IIIB. The heterodimers of (5',5') AZT and ribavirin (AZT-P-Ribavirin) and (5',5') ddI and ribavirin (ddI-P-Ribavirin) were also synthesized: AZT-P-Ribavirin inhibited HIV-1 replication, but ddI-P-Ribarvirin did not.

Inhibitory effect of 4-(2, 6-dichlorophenyl)-1, 2, 5-thiadiazol-3-yl-N-methyl, N-ethylcarbamate on replication of human immunodeficiency virus type 1 and the mechanism of action.

In the search for effective antiviral agents, we have found 4-(2, 6-dichlorophenyl)-1, 2, 5-thiadiazol-3-yl-N-methyl, N-ethylcarbamate (RD4-2025) to be a highly potent and selective inhibitor of human immunodeficiency virus type 1 (HIV-1) in vitro. The 50% effective concentration of RD4-2025 for HIV-1-induced cytopathic effect in MT-4 cells was 37 nM, yet no antiviral activity was observed against HIV-2. In HIV-1 reverse transcriptase (RT) assays, RD4-2025 inhibited both RNA-dependent and DNA-dependent DNA polymerase activities of a recombinant HIV-1 RT with 50% inhibitory concentrations of 0.11 and 3.5 microM, respectively. However, the compound did not affect the activity of human DNA polymerase alpha. Kinetic studies revealed that the inhibition was noncompetitive with respect to dGTP as the substrate and poly(C)/(dG) 12-18 as the template/primer. These results were in accordance with those of nonnucleoside RT inhibitors (NNRTIs), such as R89439 (an alpha-anilinophenylacetamide derivative) and nevirapine, indicating that RD4-2025 also belongs to the family of NNRTIs.

Different properties of wild type and drug-resistant mutants of human immunodeficiency virus type 1 reverse transcriptase in vitro.

Drug-resistant mutants of human immunodeficiency virus type 1 (HIV-1) emerge during treatment with various reverse transcriptase (RT) inhibitors in vitro and in vivo. However, the virological nature and pathogenic importance of these mutants have not been fully elucidated. In this study, we have examined HIV-1 mutants resistant to 3'-azido-3'-deoxythymidine (AZT) and nonnucleoside RT inhibitors (NNRTIs) for their infectivity, RT activity, and replication in MT-4 cells. Although the infectivity of AZT- and NNRTI-resistant mutants was similar, the RT activity of AZT-resistant mutants was much higher than that of NNRTI-resistant mutants and their wild types. Furthermore, the RT activity of NNRTI-resistant mutants was significantly lower than that of the wild types. In contrast, the replication of NNRTI-resistant mutants was found to be greater than that of AZT-resistant mutants and the wild types. When HIV-1 proviral DNA (cDNA) synthesis was examined by PCR in MT-4 cells infected with the wild type, AZT-resistant mutant, or NNRTI-resistant mutant, the PCR signal of the NNRTI-resistant mutant was found to be much higher than those of the wild type and AZT-resistant mutant. These results suggest that the drug-resistant mutants differ from their corresponding wild types not only in drug sensitivity but also in other virological properties.

Mechanism of selective inhibition of human immunodeficiency virus by ingenol triacetate.

Ingenol 3,5,20-triacetate (ITA), one of the ingenol derivatives, is a selective inhibitor of human immunodeficiency virus (HIV) replication in vitro. ITA inhibited the replication of HIV strains in MT-4 cells at concentrations of 0.051 to 0.65 microM. This concentration was approximately 10(3)-fold lower than its cytotoxic threshold. The mechanism of action of ITA is primarily attributed to the inhibition of viral adsorption to the host cells, but it is distinct from the mechanism of inhibition by other adsorption inhibitors.

Potent and specific inhibition of human immunodeficiency virus type 1 replication by 4-(2,6-dichlorophenyl)-1,2,5-thiadiazol-3-Y1 N,N-dialkylcarbamate derivatives.

4-(2,6-Dichlorophenyl)-1,2,5-thiadiazol-3-yl N,N-dialkylcarbamate (TDA) derivatives were found to be highly potent and specific inhibitors of human immunodeficiency virus type 1 (HIV-1) replication in a variety of cell cultures. The most potent congener of TDA derivatives, RD4-2024, inhibited HIV-1 replication by 50% at concentrations of 12.5 and 4.8 nM in MT-4 cells and peripheral blood mononuclear cells, respectively. These concentrations were more than 2,000- and 30,000-fold lower than its 50% cytotoxic concentrations, respectively. Although the TDA derivatives were active against 3'-azido-3'-deoxythymidine-resistant HIV-1, no antiviral activities were observed against HIV-2 and nonnucleoside reverse transcriptase inhibitor-resistant mutants of HIV-1. The TDA derivatives inhibited recombinant HIV-1 reverse transcriptase activity, depending on the template-primer used for the assay. However, they did not interact with HIV-2 reverse transcriptase. Thus, the TDA derivatives belong to the family of nonnucleoside reverse transcriptase inhibitors. Because of their potent anti-HIV-1 activities in vitro and their low levels of toxicity in mice, the TDA derivatives deserve further evaluation as candidate drugs for the treatment of patients with AIDS.

Retinal toxicity and ocular kinetics of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil in rabbits.

The intraocular penetration of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU), a new antiviral drug, after oral administration, the effects of non-toxic intravitreal doses of BV-araU, and the intraocular kinetics of BV-araU after intraocular injection were studied in rabbits. The intravitreal penetration of BV-araU after oral administration was very poor: 0.11 +/- 0.13 micrograms/ml and 0.20 +/- 0.02 microgram/ml respectively in albino and pigmented rabbits 2 h after 30 mg/kg. An intravitreal injection of 200 micrograms BV-araU caused transient electroretinographic (ERG) changes, whereas a 100-micrograms injection and intravitreal irrigation with 20 micrograms/ml BV-araU caused no ERG and histologic changes over the 4-week follow-up period. The half-life of the intravitreal concentration of BV-araU after an intravitreal injection was short (2.4 h). The results suggest that an intravitreal injection of 100 micrograms BV-araU or an intravitreal irrigating solution containing 20 micrograms/ml BV-araU is non-toxic to the retina and may be used for treatment of retinitis caused by varicella-zoster virus or herpes simplex virus type 1.

MTT colorimetric assay system for the screening of anti-orthomyxo- and anti-paramyxoviral agents.

A rapid and sensitive method was developed for screening potential antiviral agents against orthomyxo- and paramyxoviruses, using the MTT method with cell culture suspensions. The cell lines used for the assay were as follows: MDCK cells for the influenza A virus (Fluv. A), HeLa cells for the respiratory syncytial virus (RSV), and Vero cells for the measles virus (MSV). Test compounds were diluted and plated in 96-well round-bottomed microtiter plates. Trypsinized cell suspensions and viruses were added to each well, the plates were then centrifuged (700 x g, 5 min, room temperature), and incubated for several days. The MTT assay was carried out after the degeneration of virus-infected cells became evident. The optical density (OD) of formazan was determined using a computer-controlled microplate reader. With this assay system, the EC50 values of Ribavirin (used as the reference compound) were 3.7 micrograms/ml for Fluv. A, 4.5 micrograms/ml for RSV, and 12.3 micrograms/ml for MSV, respectively. These EC50 values were equivalent to those obtained using the plaque reduction assay. The confluent cell culture system was inadequate for antiviral assays against RSV and MSV when the MTT method was used, because the inhibition of formazan formation was not observed in viral-infected cells. Moreover, the suspension method is more sensitive to the cytotoxicity of antiviral agents than the confluent cell culture system.

In vivo antiviral effects of mismatched double-stranded RNA on duck hepatitis B virus.

The antiviral activity and ability of mismatched double-stranded RNA (m-dsRNA), r(I)n.r(C12-U)n, to induce interferon (IFN) were evaluated in ducks chronically infected with duck hepatitis B virus (DHBV). When m-dsRNA was administered intravenously at a single dose of 5 mg/kg, serum DHBV DNA concentrations decreased significantly for 3 days (P < 0.002). However, the DHBV DNA concentrations returned to the pretreatment levels 4 days after treatment. Inhibition of DHBV DNA replication in the liver was also observed 2 days after treatment. Serum IFN activity peaked 3 hours after administration of m-dsRNA, then rapidly declined. 2'-5' Oligo-adenylate synthetase (2'-5'AS) activity increased gradually after treatment and remained elevated for at least 48 hours. In ducks receiving m-dsRNA once daily for 7 consecutive days, serum DHBV DNA concentrations on the last day of treatment were decreased by 76 +/- 12% (P < 0.05) in ducks that received 0.2 mg of m-dsRNA per kg and by 65 +/- 12% (P < 0.05) in ducks that received 1 mg of m-dsRNA per kg. This decrease persisted for at least 2 weeks after the cessation of treatment in all ducks. These results suggest that m-dsRNA effectively inhibits DHBV replication in vivo, and that IFN induction and stimulation of 2'-5'AS activity contribute to the inhibition of DHBV replication by m-dsRNA.