Identification of thioridazine, an antipsychotic drug, as an antiglioblastoma and anticancer stem cell agent using public gene expression data.

Glioblastoma (GBM) is a common and malignant tumor with a poor prognosis. Glioblastoma stem cells (GSCs) have been reported to be involved in tumorigenesis, tumor maintenance and therapeutic resistance. Thus, to discover novel candidate therapeutic drugs for anti-GBM and anti-GSCs is an urgent need. We hypothesized that if treatment with a drug could reverse, at least in part, the gene expression signature of GBM and GSCs, this drug may have the potential to inhibit pathways essential in the formation of GBM and thereby treat GBM. Here, we collected 356 GBM gene signatures from public databases and queried the Connectivity Map. We systematically evaluated the in vitro antitumor effects of 79 drugs in GBM cell lines. Of the drugs screened, thioridazine was selected for further characterization because it has potent anti-GBM and anti-GSCs properties. When investigating the mechanisms underlying the cytocidal effects of thioridazine, we found that thioridazine induces autophagy in GBM cell lines, and upregulates AMPK activity. Moreover, LC3-II was upregulated in U87MG sphere cells treated with thioridazine. In addition, thioridazine suppressed GBM tumorigenesis and induced autophagy in vivo. We not only repurposed the antipsychotic drug thioridazine as a potent anti-GBM and anti-GSCs agent, but also provided a new strategy to search for drugs with anticancer and anticancer stem cell properties.

Endoplasmic reticulum ribosome-binding protein 1 (RRBP1) overexpression is frequently found in lung cancer patients and alleviates intracellular stress-induced apoptosis through the enhancement of GRP78.

Lung cancer is the leading cause of cancer deaths and is the most occurring malignancy worldwide. Unraveling the molecular mechanisms involved in lung tumorigenesis will greatly improve therapy. During early tumorigenesis, rapid proliferating tumor cells require increased activity of endoplasmic reticulum (ER) for protein synthesis, folding and secretion, thereby are subjected to ER stress. Ribosome-binding protein 1 (RRBP1) was originally identified as a ribosome-binding protein located on the rough ER and associated with unfolding protein response (UPR). In this report, we investigated the role of RRBP1 in lung cancer. RRBP1 was highly expressed in lung cancer tissue, as compared with adjacent normal tissues as assessed by immunohistochemistry (IHC) using lung cancer tissue array (n=87). Knockdown of RRBP1 by short-hairpin RNAs caused ER stress and significantly reduced cell viability and tumorigenicity. This effect was associated with a significant reduction in the expression of glucose-regulated protein 78 (GRP78). UPR regulator GRP78, an anti-apoptotic protein that is widely upregulated in cancer, has a critical role in chemotherapy resistance in some cancers. According to our results, cells with a higher level of RRBP1 were more resistant to ER stress. Ectopic expression of RRBP1 alleviated apoptosis that was induced by the ER-stress agent tunicamycin, 2-deoxy-D-glucose (2DG) or doxorubicin via enhancing GRP78 protein expression. A strong correlation was observed between the expression of RRBP1 and GRP78 in tumor biopsies using the database GSE10072. Our results also indicated that RRBP1 may involve in the regulation of mRNA stability of UPR components including ATF6 and GRP78. Taken together, RRBP1 could alleviate ER stress and help cancer cell survive. RRBP1 is critical for tumor cell survival, which may make it a useful target in lung cancer treatment and a candidate for the development of new targeted therapeutics.

Effects of calcium entry blocker (nicardipine) tocolysis in rhesus macaques: fetal plasma concentrations and cardiorespiratory changes.

Tocolytic doses of nicardipine, a dihydropyridine calcium entry blocker, were administered to chronically catheterized rhesus monkeys between days 128 and 132 of gestation. During periods of spontaneous uterine contractility, a 500 micrograms nicardipine bolus was injected intravenously, and this was followed by continuous infusion (6 micrograms/kg/min) to the mother for 1 hour. Uterine activity (amniotic fluid pressure) and maternal heart rate and blood pressure were monitored continuously. Paired maternal and fetal blood samples were drawn at frequent intervals to monitor pH, PO2, PCO2, and plasma nicardipine concentrations. Peak maternal nicardipine concentrations ranged from 175 to 865 ng/ml while peak fetal levels ranged from 7 to 35 ng/ml. Fetal heart rate and blood pressure were unaffected. However, fetuses became acidotic (pH 7.26 +/- 0.01 versus 7.33 +/- 0.01) and hypoxemic (PO2 16.0 +/- 3.2 versus 24.5 +/- 2.0 mm Hg) after maternal nicardipine treatment (p less than 0.01). Despite the fact that maternal nicardipine treatment exerted a significant tocolytic effect, the undesirable fetal side effects are of concern and deserve further investigation.

Ketorolac tromethamine absorption, distribution, metabolism, excretion, and pharmacokinetics in animals and humans.

Ketorolac tromethamine (KT), a potent non-narcotic analgesic, with cyclooxygenase inhibitory activity, was administered (14C-labeled and unlabeled) intravenously (iv), orally (po), and intramuscularly (im) in solution to humans, cynomolgus monkeys, rabbits, rats, and mice. KT was absorbed rapidly (Tmax less than 1.0 hr) and efficiently (greater than 87%) following po and im doses in all species. The plasma half-life of ketorolac (K) ranged from 1.1 hr (rabbits) to 6.0 hr (humans). The protein binding of K ranged from 72.0% (mouse) to 99.2% (humans). Linear pharmacokinetics of K was observed in the mouse after single oral doses of KT ranging from 0.25 to 16 mg/kg. Radioactivity was excreted predominantly into urine, ranging from 78.9% (mouse) to 102% (monkey) following iv doses. The dose was excreted into urine primarily as K conjugates, K, and p-hydroxy-K in humans. The monkey was similar to humans with respect to kinetics, but did not form the p-hydroxy metabolite. The rabbit was unusual in that it exhibited substantial presystemic metabolism (50%). The rat excreted a much higher percentage of radioactivity into the feces and formed an additional unidentified metabolite. The most comparable species with respect to humans metabolically was the mouse. The metabolism and excretion of K was similar following iv, po, and im doses within each species studied.

Capillary column gas chromatographic method using electron-capture detection for the simultaneous determination of nicardipine and its pyridine metabolite II in plasma.

A rapid, specific and direct method based on capillary column gas chromatography with electron-capture detection is described for the simultaneous determination of nicardipine, a new calcium antagonist, and its pyridine metabolite II in human plasma. In this method, the nicardipine, its pyridine metabolite II and internal standard are extracted from the plasma and then partially purified by acid-base partitioning prior to the final injection onto the capillary column gas chromatograph for quantification by means of an electron-capture detector. The quantification limit of the method is 1 ng/ml of plasma for both nicardipine and its pyridine metabolite II. The coefficients of variation for nicardipine and the pyridine metabolite II at concentrations of 1-50 ng/ml are less than 7% and less than 9% (n = 4), respectively. The method has been validated against a previously developed high-performance liquid chromatographic method (sensitivity 5 ng/ml).

A high-performance liquid chromatographic method for the simultaneous determination of nicardipine and its pyridine metabolite II in plasma.

A rapid and specific method in which reverse-phase high-performance liquid chromatography (HPLC) with UV detection was used for the simultaneous determination of nicardipine and its pyridine metabolite II in human plasma is described. Nicardipine, its pyridine metabolite II, and the internal standard were extracted from plasma and partially purified by acid-base partitioning. Final purification and quantitation were achieved by HPLC by using a reverse-phase column and a UV detector (254 nm). The extraction efficiencies for nicardipine and its pyridine metabolite II from 1 mL of plasma were 77.4 and 81.1%, respectively. The sensitivity of the assay was 5 ng/mL for both nicardipine and its pyridine metabolite II, and the linear concentration range of the assay was 5-150 ng/mL for both compounds. The low coefficients of variation (less than or equal to 5%) for samples spiked with nicardipine and its pyridine metabolite II in this concentration range demonstrate good reliability and reproducibility of the assay. The HPLC procedure has been validated by comparison with a GC-electron-capture detection (ECD) procedure, which gives the combined concentration of nicardipine-its pyridine metabolite II (total) and with an HPLC/GC-ECD procedure, which gives the concentration of its pyridine metabolite II. All three methods, which were developed in our laboratory, were used to analyze nicardipine and its pyridine metabolite II in specimens of plasma from subjects treated with nicardipine hydrochloride. Good correlations were found for concentrations of nicardipine, its pyridine metabolite II, and nicardipine plus the metabolite determined by these three procedures. The HPLC procedure is suitable for use in pharmacokinetic studies following administration of nicardipine hydrochloride to humans.

Transformation of arachidonate into 6-oxoprostaglandin F1 alpha, thromboxane B2 and prostaglandin E2 by sheep lung microsomal fraction.

In the presence of haemoglobin and isoproterenol, the microsomal fraction of sheep lung catalysed the conversion of arachidonate predominantly into thromboxane B2 and to a lesser extent into 6-oxoprostaglandin F1alpha. Very little prostaglandin E2 and prostaglandin F2alpha were formed. If reduced glutathione was added in combination with haemoglobin and isoproterenol, the synthesis of prostaglandin E2 was favoured over that of thromboxane B2 and 6-oxoprostaglandin F1alpha. The identities of these products were confirmed by t.l.c. and by combined g.l.c.-mass spectrometry. These results indicate that microsomal fraction of sheep lung possesses active prostaglandin synthase, prostacyclin synthase and thromboxane synthase activities.

Hydroxylated metabolies of R,S-1-(tetrahydro-2-furanyl)-5-fluorouracil (Ftorafur) in rats and rabbits.

Two hydroxylated metabolies (M1 and M2) have been isolated from rabbit urine after administration of Ftorafur (FT). The structures of 3'-OH-FT and 4'-OH-FT were assigned to M1 and M2, respectively. A reverse-phase high performance liquid chromatography assay was developed for jeasuring FT, M1, M2, and 5-fluorouracil (FU) plasma levels. M1, M2, and FU were present in rabbit and rat plasma in greatly varying concentrations after FT administration. Pharmacokinetic studies suggest that FU formation proceeds via metabolic intermediate(s) and that the extent of FT activation is variable. A horse liver thymidine phosphorylase ,reparation capable of catalyzing the conversion of beta-ribo-2'-deoxy-5-fluorouracil to FU was inactive against FT and M1. However, 20% of M2 was converted to FU by this enzume, which suggests that the urinary metabolite M2 consisted of a mixture of enantiomers with 20% present in the natural beta-D configuration. The stereochemistry of M1 remains unknown. Hydroxylation of FT to beta-D-4'-OH-FT and subsequent cleavage to FU by thymidine phosphorylase represents one possible activation mechanism of FT to FU. ,owever, lack of correlation between plasma levels of M2 and FU indicates that this mode of metabolic activation may account for only part of the overall activation of FT in vivo.

Determination of ftorafur and 5-fluorouracil levels in plasma and urine.

A gas chromatographic method was developed for ftorafur (Ft) detection in plasma and urine with a sensitivity of 1 mug/ml. Specific determination of its metabolite 5-fluorouracil (FU) with a sensitivity of 1 ng/ml was achieved by column chromatographic separation from Ft and subsequent gas chromatography-mass spectrometry of bis-silyl-FU in the selected ion mode (GC-MS-SIM) using bis-15N-FU as internal standard. Intravenous injections of 2-14C-Ft and 2',5'-14C-Ft were given to rats and rabbits respectively, and plasma and urine were analyzed for Ft, and 14C activity. Unchanged Ft accounted for most of the 14C activity in plasma, while FU concentrations were below 0.15% and 0.4% relative to Ft concentrations in the rabbit and the rat, respectively. 30-60% of the urinary 14C activity was unchanged Ft and less than 0.2% FU. The significance or low FU levels is discussed in view of the hypothesis that Ft acts as a transport form of its metabolite FU.