bluuebunny/crossref_metadata_2025_split huggingface dataset:
Metabolism and pharmacokinetics of the cyclin-dependent kinase inhibitorR-roscovitine in the mouse
Authors: Nutley Bernard P. , Raynaud Florence I. , Wilson Stuart C. , Fischer Peter M. , Hayes Angela , Goddard Phyllis M. , McClue Steven J. , Jarman Michael , Lane David P. , Workman Paul 
Published: June 2022

AbstractR-roscovitine (seliciclib, CYC202) is a cyclin-dependent kinase inhibitor currently in phase II clinical trials in patients with cancer. Here, we describe its mouse metabolism and pharmacokinetics as well as the identification of the principal metabolites in hepatic microsomes, plasma, and urine. Following microsomal incubation of R-roscovitine at 10 μg/mL (28 μmol/L) for 60 minutes, 86.7% of the parent drug was metabolized and 60% of this loss was due to formation of one particular metabolite. This was identified as the carboxylic acid resulting from oxidation of the hydroxymethyl group of the amino alcohol substituent at C2 of the purine core present in R-roscovitine. Identification was confirmed by chemical synthesis and comparison of an authentic sample of the R-roscovitine-derived carboxylate metabolite (COOH-R-roscovitine). Other minor metabolites were identified as C8-oxo-R-roscovitine and N9-desisopropyl-R-roscovitine; these accounted for 4.9% and 2.6% of the parent, respectively. The same metabolic pattern was observed in vivo, with a 4.5-fold lower AUC∞ for R-roscovitine (38μmol/L/h) than for COOH-R-roscovitine (174 μmol/L/h). Excretion of R-roscovitine in the urine up to 24 hours post-dosing accounted for an average of only 0.02% of the administered dose of 50 mg/kg, whereas COOH-R-roscovitine represented 65% to 68% of the dose irrespective of the route of administration (i.v., i.p., or p.o.). A partially deuterated derivative (R-roscovitine-d9) was synthesized to investigate if formation of COOH-R-roscovitine could be inhibited by replacement of metabolically labile protons with deuterium. After 60 minutes of incubation of R-roscovitine-d9 or R-roscovitine with mouse liver microsomes, formation of COOH-R-roscovitine-d9 was decreased by ∼24% compared with the production of COOH-R-roscovitine. In addition, the levels of R-roscovitine-d9 remaining were 33% higher than those of R-roscovitine. However, formation of several minor R-roscovitine metabolites was enhanced with R-roscovitine-d9, suggesting that metabolic switching from the major carbinol oxidation pathway had occurred. Synthetic COOH-R-roscovitine and C8-oxo-R-roscovitine were tested in functional cyclin-dependent kinase assays and shown to be less active than R-roscovitine, confirming that these metabolic reactions are deactivation pathways.
Role of phorbol ester localization in determining protein kinase C or RasGRP3 translocation: Real-time analysis using fluorescent ligands and proteins
Authors: Braun Derek C. , Cao Yeyu , Wang Shaomeng , Garfield Susan H. , Min Hur Gang , Blumberg Peter M. 
Published: June 2022

Abstract
The diacylglycerol signaling pathway, involving protein kinase C (PKC) and RasGRP, is a promising therapeutic target for both cancer and other indications. The phorbol esters, ultrapotent diacylglycerol analogues, bind to and activate PKC and RasGRP. Here, using fluorescent phorbol esters and complementary fluorescent PKC and RasGRP constructs, we determined the localization of the phorbol ester as a function of time after addition and how the resultant PKC or RasGRP3 translocation related to ligand localization. For these studies, we prepared fluorescently labeled phorbol esters of varying lipophilicities based on the BODIPY FL (green) or BODIPY 581/591 (red) fluorophores, and by using fusion constructs of green fluorescent protein or DsRed with PKC isoforms or RasGRP3 expressed in Chinese hamster ovary cells, we simultaneously compared the kinetics and pattern of localization of PKC or RasGRP3 with that of the fluorescent red or green phorbol esters. Binding assays showed that the fluorescent derivatives were potent ligands. Uptake followed a one-compartment pharmacokinetic model with a half-time of minutes to hours, depending on the ligand, and all of the fluorescent phorbol esters localized primarily to intracellular membranes, with little plasma membrane localization. The fluorescent phorbol esters induced translocation of and generally colocalized with PKCδ or RasGRP3. However, PKCα and, initially, PKCδ, translocated to the plasma membrane, in which little phorbol ester accumulated. The findings argue that the rate of uptake of phorbol esters influences the subsequent pattern of PKCδ translocation, and that the specificity for PKCα translocation is dominated by factors other than the localization of the ligand.
8-Amino-adenosine is a potential therapeutic agent for multiple myeloma
Authors: Krett Nancy L. , Davies Katharine M. , Ayres Mary , Ma Chunguang , Nabhan Chadi , Gandhi Varsha , Rosen Steven T. 
Published: June 2022

Abstract
Multiple myeloma (MM) is a malignancy of clonal B-cells that accounts for 10% of all hematologic malignancies. We have shown previously that a novel purine analogue, 8-chloro-adenosine, has significant activity for MM in preclinical studies. Objective: Using MM cell lines, we investigated the molecular mechanism of related congener of adenosine, 8-amino-adenosine (8-NH2-Ado). Methods: We employed biological and biochemical assays in MM cell lines to evaluate the clinical potential of 8-NH2-Ado. Results: In MM cell lines both sensitive and resistant to conventional chemotherapies, 8-NH2-Ado is cytotoxic, with IC50 ranging from 300 nmol/L to 3 μmol/L. A mouse leukemic cell line lacking adenosine kinase activity was resistant to 8-NH2-Ado, indicating that phosphorylation of 8-NH2-Ado to its triphosphate form is required for cytotoxicity. A 4-hour incubation of MM cells with 10 μmol/L analogue resulted in an accumulation of &gt;7 mmol/L 8-NH2-ATP with a parallel decline in the endogenous ATP levels. Accumulation of 8-NH2-ATP was dependent on both exogenous concentration of 8-NH2-Ado and incubation time. The accumulation of 8-NH2-ATP was accompanied by a decrease in both RNA and DNA synthesis. The mechanism of 8-NH2-Ado-mediated cytotoxicity was due to apoptosis as measured by an increase in Annexin V binding, a decrease in mitochondrial membrane potential, an increase in caspase activity, cleavage of caspase substrates, and an increase in cells with a sub-G1 DNA content. Conclusion: Based on these results, we conclude that 8-NH2-Ado may hold great potential as a therapeutic agent for the treatment of MM.
Screening a combinatorial peptide library to develop a human glandular kallikrein 2–activated prodrug as targeted therapy for prostate cancer
Authors: Janssen Samuel , Jakobsen Carsten M. , Rosen D. Marc , Ricklis Rebecca M. , Reineke Ulrich , Christensen Soeren B. , Lilja Hans , Denmeade Samuel R. 
Published: June 2022

AbstractObjective: Prostate cancer cells secrete the unique protease human glandular kallikrein 2 (hK2) that represents a target for proteolytic activation of cytotoxic prodrugs. The objective of this study was to identify hK2-selective peptide substrates that could be coupled to a cytotoxic analogue of thapsigargin, a potent inhibitor of the sarcoplasmic/endoplasmic reticulum calcium ATPase pump that induces cell proliferation–independent apoptosis through dysregulation of intracellular calcium levels. Methods: To identify peptide sequence requirements for hK2, a combination of membrane-bound peptides (SPOT analysis) and combinatorial chemistry using fluorescence-quenched peptide substrates was used. Peptide substrates were then coupled to 8-O-(12[l-leucinoylamino]dodecanoyl)-8-O-debutanoylthapsigargin (L12ADT), a potent analogue of thapsigargin, to produce a prodrug that was then characterized for hK2 hydrolysis, plasma stability, and in vitro cytotoxicity. Results: Both techniques indicated that a peptide with two arginines NH2-terminal of the scissile bond produced the highest rates of hydrolysis. A lead peptide substrate with the sequence Gly-Lys-Ala-Phe-Arg-Arg (GKAFRR) was hydrolyzed by hK2 with a Km of 26.5 μmol/L, kcat of 1.09 s−1, and a kcat/Km ratio of 41,132 s−1 mol/L−1. The GKAFRR-L12ADT prodrug was rapidly hydrolyzed by hK2 and was stable in plasma, whereas the GKAFRR-L peptide substrate was unstable in human plasma. The hK2-activated thapsigargin prodrug was not activated by cathepsin B, cathepsin D, and urokinase but was an excellent substrate for plasmin. The GKAFRR-L12ADT was selectively cytotoxic in vitro to cancer cells in the presence of enzymatically active hK2. Conclusion: The hK2-activated thapsigargin prodrug represents potential novel targeted therapy for prostate cancer.
The role of nuclear Y-box binding protein 1 as a global marker in drug resistance
Authors: Kuwano Michihiko , Oda Yoshinao , Izumi Hiroto , Yang Song-Ju , Uchiumi Takeshi , Iwamoto Yukihide , Toi Masakazu , Fujii Teruhiko , Yamana Hideaki , Kinoshita Hisafumi , Kamura Toshiharu , Tsuneyoshi Masazumi , Yasumoto Kosei , Kohno Kimitoshi 
Published: June 2022

AbstractGene expression can be regulated by nuclear factors at the transcriptional level. Many such factors regulate MDR1 gene expression, but what are the sequence elements and transcription factors that control the basal and inducible expression of this gene? The general principles through which transcription factors participate in drug resistance are now beginning to be understood. Here, we review the factors involved in the transcriptional regulation of the MDR1 gene. In particular, we focus on the transcription factor Y-box binding protein 1 and discuss the possible links between Y-box binding protein 1 expression and drug resistance in cancer, which are mediated by the transmembrane P-glycoprotein or non–P-glycoprotein.
Akt up-regulation increases resistance to microtubule-directed chemotherapeutic agents through mammalian target of rapamycin
Authors: VanderWeele David J. , Zhou Rixin , Rudin Charles M. 
Published: June 2022

Abstract
Chemotherapeutic agents induce apoptosis in cancer cells through effects on multiple intracellular targets. Recent observations suggest that a consistent cellular response to chemotherapeutic agents of disparate classes is down-regulation of glycolytic metabolism. Inhibition of glycolytic activity has been linked to apoptotic induction in several models. The serine/threonine kinase Akt (protein kinase B) promotes both glycolytic metabolism and survival, and these functions have been shown to be linked. Because of its key role in both glycolysis and survival, we examined the function of Akt in the cellular response to cytotoxic agents. Following exposure to any of several chemotherapeutic agents, an initial up-regulation in endogenous Akt activity is rapidly suppressed. Using cells containing constitutively active myristoylated Akt, dominant-negative kinase-dead Akt, or an empty vector control, we show here that Akt activation markedly increases resistance to microtubule-directed agents, including vincristine, colchicine, and paclitaxel. Akt also maintains increased glycolytic rate in response to antimicrotubule treatment. Rapamycin inhibits Akt-mediated maintenance of glycolysis and therapeutic resistance, indicating that these effects are dependent on mammalian target of rapamycin (mTOR). Furthermore, an activated mTOR mutant confers resistance to antimicrotubule agents. Taken together, these observations suggest that activation of the Akt-mTOR signaling pathway can augment glucose utilization and promote resistance to chemotherapeutic agents that do not directly target metabolic regulation. These data provide insight into potentially synergistic combinations of anticancer therapies.
The ex vivo characterization of XR5944 (MLN944) against a panel of human clinical tumor samples
Authors: Di Nicolantonio Federica , Knight Louise A. , Whitehouse Pauline A. , Mercer Stuart J. , Sharma Sanjay , Charlton Peter A. , Norris David , Cree Ian A. 
Published: June 2022

Abstract
XR5944 (MLN944) is a novel DNA targeting agent with potent antitumor activity, both in vitro and in vivo, against several murine and human tumor models. We have used an ATP-tumor chemosensitivity assay to assess the ex vivo sensitivity of a variety of solid tumors (n = 90) and a CCRF-CEM leukemia cell line selected with XR5944. Differences in gene expression between the parental CCRF-CEM and the resistant subline were investigated by quantitative reverse transcription-PCR. Immunohistochemistry for topoisomerases I and IIα and multidrug resistance (MDR1) protein was done on those tumors for which tissue was available (n = 32). The CCRF-CEM XR5944 line showed increased mRNA levels of MDR1, major vault protein, and MDR-associated protein 1 compared with the parental line, whereas the expression of topoisomerases I, IIα, and IIβ was essentially unchanged, suggesting that XR5944 is susceptible to MDR mechanisms. The median IC90 and IC50 values for XR5944 in tumor-derived cells were 68 and 26 nmol/L, respectively, 6-fold greater than in resistant cell lines. XR5944 was 40- to 300-fold more potent than the other cytotoxics tested, such as doxorubicin, topotecan, and paclitaxel. Breast and gynecologic malignancies were most sensitive to XR5944, whereas gastrointestinal tumors showed greater resistance. A positive correlation (r = 0.68; P &lt; 0.0001) was found between the IC50 values of XR5944 and P-glycoprotein/MDR1 staining but not with either topoisomerase I or IIα immunohistochemistry index. These data support the rapid introduction of XR5944 to clinical trials and suggest that it may be effective against a broad spectrum of tumor types, especially ovarian and breast cancer.
KRN633: A selective inhibitor of vascular endothelial growth factor receptor-2 tyrosine kinase that suppresses tumor angiogenesis and growth
Authors: Nakamura Kazuhide , Yamamoto Atsushi , Kamishohara Masaru , Takahashi Kazumi , Taguchi Eri , Miura Toru , Kubo Kazuo , Shibuya Masabumi , Isoe Toshiyuki 
Published: June 2022

Abstract
Vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 play a central role in angiogenesis, which is necessary for solid tumors to expand and metastasize. Specific inhibitors of VEGFR-2 tyrosine kinase are therefore thought to be useful for treating cancer. We showed that the quinazoline urea derivative KRN633 inhibited tyrosine phosphorylation of VEGFR-2 (IC50 = 1.16 nmol/L) in human umbilical vein endothelial cells. Selectivity profiling with recombinant tyrosine kinases showed that KRN633 was highly selective for VEGFR-1, -2, and -3. KRN633 also blocked the activation of mitogen-activated protein kinases by VEGF, along with human umbilical vein endothelial cell proliferation and tube formation. The propagation of various cancer cell lines in vitro was not inhibited by KRN633. However, p.o. administration of KRN633 inhibited tumor growth in several in vivo tumor xenograft models with diverse tissue origins, including lung, colon, and prostate, in athymic mice and rats. KRN633 also caused the regression of some well-established tumors and those that had regrown after the cessation of treatment. In these models, the trough serum concentration of KRN633 had a more significant effect than the maximum serum concentration on antitumor activity. KRN633 was well tolerated and had no significant effects on body weight or the general health of the animals. Histologic analysis of tumor xenografts treated with KRN633 revealed a reduction in the number of endothelial cells in non-necrotic areas and a decrease in vascular permeability. These data suggest that KRN633 might be useful in the treatment of solid tumors and other diseases that depend on pathologic angiogenesis.
Carotenoids activate the antioxidant response element transcription system
Authors: Ben-Dor Anat , Steiner Michael , Gheber Larisa , Danilenko Michael , Dubi Noga , Linnewiel Karin , Zick Anat , Sharoni Yoav , Levy Joseph 
Published: June 2022

Abstract
Epidemiologic studies have found an inverse association between consumption of tomato products and the risk of certain types of cancers. However, the mechanisms underlying this relationship are not completely understood. One mechanism that has been suggested is induction of phase II detoxification enzymes. Expression of phase II enzymes is regulated by the antioxidant response element (ARE) and the transcription factor Nrf2 (nuclear factor E2-related factor 2). In this study, we determined the role of this transcription system in the induction of phase II enzymes by carotenoids. We found that in transiently transfected cancer cells, lycopene transactivated the expression of reporter genes fused with ARE sequences. Other carotenoids such as phytoene, phytofluene, β-carotene, and astaxanthin had a much smaller effect. An increase in protein as well as mRNA levels of the phase II enzymes NAD(P)H:quinone oxidoreductase and γ-glutamylcysteine synthetase was observed in nontransfected cells after carotenoid treatment. Ethanolic extract of lycopene containing unidentified hydrophilic derivatives of the carotenoid activated ARE with similar potency to lycopene. The potency of the carotenoids in ARE activation did not correlate with their effect on intracellular reactive oxygen species and reduced glutathione level, which may indicate that ARE activation is not solely related to their antioxidant activity. Nrf2, which is found predominantly in the cytoplasm of control cells, translocated to the nucleus after carotenoid treatment. Interestingly, part of the translocated Nrf2 colocalized with the promyelocytic leukemia protein in the promyelocytic leukemia nuclear bodies. The increase in phase II enzymes was abolished by a dominant-negative Nrf2, suggesting that carotenoid induction of these proteins depends on a functional Nrf2 and the ARE transcription system.
Staurosporine induces apoptosis of melanoma by both caspase-dependent and -independent apoptotic pathways
Authors: Zhang Xu Dong , Gillespie Susan K. , Hersey Peter 
Published: June 2022

Abstract
Staurosporine has long been used in vitro as an initiator of apoptosis in many different cell types, but the mechanism involved remains poorly understood. In the present study, we have examined the apoptosis-inducing potential of staurosporine in cultured melanoma cell lines and dissected the staurosporine-induced apoptotic signaling pathway. We report that although staurosporine activated Bax and the mitochondrial caspase-dependent apoptotic pathway, it also induced apoptosis of melanoma by caspase-independent pathways. The caspase-dependent apoptotic pathway was activated relatively soon after exposure to staurosporine and was associated with release of cytochrome c and Smac/DIABLO from mitochondria and cleavage of poly(ADP-ribose) polymerase and inhibitor of caspase-activated DNase. This pathway was inhibitable by broad caspase inhibitors. A second apoptotic pathway that appeared to be involved in late apoptotic events was caspase independent in that inhibitors of caspases did not prevent the late onset of apoptosis. Overexpression of Bcl-2 inhibited the early onset of apoptosis but not the later, caspase-independent pathway. Apoptosis-inducing factor may be responsible for the late apoptotic execution in that its translocation from mitochondria into the nucleus coincided with the late onset of apoptosis and could not be inhibited by either a pan-caspase inhibitor or overexpression of Bcl-2. Our results indicate that staurosporine is able to bypass resistance of melanoma cells to mitochondrial caspase-dependent apoptotic pathways; hence, derivatives of staurosporine may warrant further evaluation either alone or with other apoptosis-inducing agents.
Differential effect of cadmium on cholinephosphotransferase activity in normal and cancerous human mammary epithelial cell lines
Authors: Roy Somdutta Sinha , Mukherjee Shyamali , Mukhopadhyay Sutapa , Das Salil K. 
Published: June 2022

Abstract
Cadmium (Cd) is an ubiquitous environmental carcinogen. Membrane phospholipids as well as fatty acid profile of membrane phospholipids are known to be altered in tumorigenicity and malignancy. Synthesis of cellular phosphatidylcholine (PC) has been used as a marker for membrane proliferation in the neoplastic mammary gland tissue. Cholinephosphotransferase (CPT), the terminal enzyme in de novo synthesis of PC, has an important role in regulating the acyl group of PC in mammalian cells. Our previous studies have shown that CPT is expressed differentially in the normal and cancerous mammary epithelial cell lines. In this study, we examined the effect of cadmium on CPT activity using normal (MCF-12A and MCF-12F) and cancerous (MCF-7, BT-549, and 11-9-1-4) human mammary epithelial cell lines. There was no consistent pattern of CPT activity in response to different doses of cadmium. The activity did not show a time-dependent variation at 5 μm concentration, except in MCF-7 and 11-9-1-4. CPT gene expression increased with cadmium as evident from slot blots. Mutation in the nucleotide sequence was also observed as the result of cadmium but this did not result into amino acid sequence changes.
Phosphorylation of extracellular signal-regulated kinase 1 and 2, protein kinase B, and signal transducer and activator of transcription 3 are differently inhibited by an epidermal growth factor receptor inhibitor, EKB-569, in tumor cells and normal human keratinocytes
Authors: Nunes Maria , Shi Celine , Greenberger Lee M. 
Published: June 2022

Abstract
EKB-569 is an irreversible inhibitor of epidermal growth factor receptor (EGF-R) tyrosine kinase. It inhibits EGF-induced phosphorylation of EGF-R and the growth of tumors that overexpress EGF-R in animal models. In clinical trials, EKB-569 and all other EGF-R inhibitors cause skin rashes. To understand the latter phenomenon, the effect of EKB-569 on EGF-R as well as downstream signaling to phosphoinositide 3-kinase-protein kinase B (AKT), extracellular signal-regulated kinase 1 and 2 (ERK1/2), or signal transducer and activator of transcription 3 (STAT3) pathways were compared in tumor cell lines and normal human keratinocytes (NHEK) grown in tissue culture. Tumor cell lines that have high (A431 epidermoid and MDA-468 breast carcinomas) and low (MCF-7 breast carcinoma) expression of EGF-R were used. NHEK cells express at least 15-fold less EGF-R than A431 cells. EKB-569 was a potent inhibitor of proliferation in NHEK, A431, and MDA-468 cells (IC50 = 61, 125, and 260 nm, respectively) but not MCF-7 cells (IC50 = 3600 nm). EKB-569 was also a potent inhibitor of EGF-induced phosphorylated EGF-R (pEGF-R) in A431 and NHEK cells (IC50 = 20–80 nm). The reduction in pEGF-R paralleled inhibition of phosphotyrosine-705 STAT3, while the inhibition of phosphorylated AKT and phosphorylated ERK1/2 occurred at higher concentrations of EKB-569 (75–500 nm) in both A431 and NHEK cells. The effects were specific because EKB-569 did not inhibit the nuclear factor-κB pathway. It is proposed that skin toxicity associated with EKB-569 is due to inhibition of EGF-R signaling. Downstream signal transduction markers, particularly the activation status of STAT3, may be useful surrogate markers to guide clinical development of EGF-R inhibitors.