Chronic wasting disease (CWD), a transmissible prion disease that affects elk and deer, poses new challenges to animal and human health. Although the transmission of CWD to humans has not been ...proven, it remains a possibility. If this were to occur, it is important to know whether the “acquired” human prion disease would show a phenotype including the scrapie prion protein (PrPSc) features that differ from those associated with human sporadic prion disease. In this study, we have compared the pathological profiles and PrPSc characteristics in brains of CWD-affected elk and deer with those in subjects with sporadic Creutzfeldt-Jakob disease (CJD), as well as CJD-affected subjects who might have been exposed to CWD, using histopathology, immunohistochemistry, immunoblotting, conformation stability assay, and N-terminal protein sequencing. Spongiform changes and intense PrPSc staining were present in several brain regions of CWD-affected animals. Immunoblotting revealed three proteinase K (PK)-resistant bands in CWD, representing different glycoforms of PrPSc. The unglycosylated PK-resistant PrPSc of CWD migrated at 21 kDa with an electrophoretic mobility similar to that of type 1 human PrPSc present in sporadic CJD affecting subjects homozygous for methionine at codon 129 (sCJDMM1). N-terminal sequencing showed that the PK cleavage site of PrPSc in CWD occurred at residues 82 and 78, similar to that of PrPSc in sCJDMM1. Conformation stability assay also showed no significant difference between elk CWD PrPSc and the PrPSc species associated with sCJDMM1. However, there was a major difference in glycoform ratio of PrPSc between CWD and sCJDMM1 affecting both subjects potentially exposed to CWD and non-exposed subjects. Moreover, PrPSc of CWD exhibited a distinct constellation of glycoforms distinguishable from that of sCJDMM1 in two-dimensional immunoblots. These findings underline the importance of detailed PrPSc characterization in trying to detect novel forms of acquired prion disease.
Bortezomib reversibly inhibits 26S proteasomal degradation, interferes with NF-κB, and exhibits antitumor activity in human malignancies. Zinc finger protein Sp1 transactivates DNMT1 gene in mice and ...is functionally regulated through protein abundance, posttranslational modifications (ie, ubiquitination), or interaction with other transcription factors (ie, NF-κB). We hypothesize that inhibition of proteasomal degradation and Sp1/NF-κB–mediated transactivation may impair aberrant DNA methyltransferase activity. We show here that, in addition to inducing accumulation of polyubiquitinated proteins and abolishment of NF-κB activities, bortezomib decreases Sp1 protein levels, disrupts the physical interaction of Sp1/NF-κB, and prevents binding of the Sp1/NF-κB complex to the DNMT1 gene promoter. Abrogation of Sp1/NF-κB complex by bortezomib causes transcriptional repression of DNMT1 gene and down-regulation of DNMT1 protein, which in turn induces global DNA hypomethylation in vitro and in vivo and re-expression of epigenetically silenced genes in human cancer cells. The involvement of Sp1/NF-κB in DNMT1 regulation is further demonstrated by the observation that Sp1 knockdown using mithramycin A or shRNA decreases DNMT1 protein levels, which instead are increased by Sp1 or NF-κB overexpression. Our results unveil the Sp1/NF-κB pathway as a modulator of DNA methyltransferase activity in human cancer and identify bortezomib as a novel epigenetic-targeting drug.
Aggressive Chronic lymphocytic leukemia (CLL) exhibits reduced miR-29b with poor outcome. Strategically increasing miR-29b to reprogram CLL cells provides therapeutic potential. However, miRs are ...sensitive to degradation in circulation and encapsulation by cationic lipocomplex leads non-specific delivery, thus causing limited uptake in CLL cells. To overcome these limitations, we designed miR29b-encapsuled immunoliposome (2A2-miR-29b-ILP) targeting ROR1, a receptor tyrosine kinase expressed in leukemic but not normal B cells. The 2A2-ILP enhanced miR-29b uptake by >20-fold compared to free miR29b and 3-fold to IgG-ILP control. Interestingly, 2A2-miR-29b-ILP treatment did not directly affect cellular viability nor BCL2 pathways, but significantly decreased the global DNA methylation through downregulation of DNMTs and SP1 in primary CLL cells and a human CLL cell line OSU-CLL. (n=3 CLL, DNMT1: p= 0.0115; DNMT3a: p=0.0231, SP1; p=0.0031). Consistent with the downregulation of DNMTs, decreased global DNA methylation was observed in OSU-CLL cell line one week after treatment with 2A2-ILP-miR-29b (n=3, p=0.0003). Furthermore, using a double transgenic Eμ-TCL-1-ROR1 mouse model expressing human ROR1 (hROR1) on leukemic B cells, we further demonstrated the therapeutic benefit of enhanced survival with 2A2-miR-29b-ILP via cellular reprograming by downregulation of DNMT1 and DNMT3a in-vivo. Using the hROR1+CD19+CD5+ leukemic cell adoptive transfer model, we observed significant in-vivo efficacy of 2A2-miR-29b-ILP (also referred as 2A2-29b) formulation, compared to scramble control (referred as 2A2-SC), associated with a) decreased number of circulating leukemic B220+CD5+ cells (58±3% decrease in 2A2-29b treatment, p=0.0184; 2A2-29b: n=9; 2A2-SC: n=7) b) reduced splenomegaly (53±2% decrease in 2A2-29b treatment, p=0.0002, n=4) c) with extended survival (p=0.0075, 2A2-29b: n=9; 2A2-SC: n=7). Moreover, our RNAseq profiling of hROR1xTCL1 splenocyte implied reprogramming of cell cycle by miR-29b treatment relieving the leukemic progression. Gene expression of miR-29b treated leukemic splenocytes revealed 128 of 233 differentially expressed genes to be associated with cell growth and proliferation. The in-vivo miR29b-treated splenocytes exhibited compromised cell cycle, associated with decreased SP1 and increased p21 protein expression in our immunoblotting analysis. Importantly, SP1-knockdown resulted in significant decrease in miR29b mediated cell cycle arrest implicating SP1 is involved in miR-29b cell cycle arrest/suppression. In conclusion, 2A2-ILP effectively delivered functional miR-29b, resulting in DNMTs downregulation, reduction of DNA methylation and anti-leukemic activity. These studies form a basis of leukemic cell targeted delivery of miR29b as a promising therapeutic approach for CLL without compromising the ROR1-ve normal B cell counterparts.
Chi-Ling Chiang is a recipient of Pelotonia Graduate Student Fellowship. This work was supported by NIH Grants
Byrd:Acerta Pharma: Research Funding; Janssen: Research Funding; Genentech: Research Funding; Pharmacyclics: Research Funding; The Ohio State University: Patents & Royalties: OSU-2S.
A disubstituted polyacetylene with naphthalene pendant (–{(C
6H
5)CC(CH
2)
3ONap}
n
–), Nap=1-napthyl; (
1) was synthesized. Upon photoexcitation, THF solutions of (
1) emitted strong blue light of ...460 nm, whose quantum efficiency is comparable to that of poly(1-phenyl-1-octyne), a well-known highly emissive disubstituted polyacetylene. Little shift was observed in the luminescence spectra of the thin solid films of (
1). An electroluminescence device with a configuration of ITO/(
1):PVK/bathocuproine/Alq
3/LiF/Al was constructed, which emitted blue light of 468 nm with a maximum brightness of 955 cd/m
2 and a power efficiency of 0.18 lm/W.
In this study, we characterized the pharmacokinetics of OSU-2S, a fingolimod-derived, non-immunosuppressive phosphatase activator, in mice, rats, and dogs, as well as tolerability and food effects in ...dogs. Across all species tested, plasma protein binding for OSU-2S was > 99.5%, and metabolic stability and hepatic intrinsic clearance were in the moderate range. OSU-2S did not significantly modulate CYP enzyme activity up until 50 μM, and Caco-2 data suggested low permeability with active efflux at 2 μM. Apparent oral bioavailability in mice was 16% and 69% at 10 and 50 mg/kg, respectively. In rats, bioavailability was 24%, 35%, and 28% at 10, 30, and 100 mg/kg, respectively, while brain/plasma ratio was 36 at 6-h post-dose at 30 mg/kg. In dogs, OSU-2S was well tolerated with oral capsule bioavailability of 27.5%. Plasma OSU-2S exposures increased proportionally over a 2.5–20 mg/kg dose range. After 4 weeks of 3 times weekly, oral administration (20 mg/kg), plasma AUC
last
(26.1 μM*h), and
C
max
(0.899 μM) were nearly 2-fold greater than those after 1 week of dosing, and no food effects were observed. The elimination half-life (29.7 h), clearance (22.9 mL/min/kg), and plasma concentrations of repeated oral doses support a 3-times weekly dosing schedule in dogs. No significant CBC, serum biochemical, or histopathological changes were observed. OSU-2S has favorable oral PK properties similar to fingolimod in rodents and dogs and is well tolerated in healthy animals. This work supports establishing trials of OSU-2S efficacy in dogs with spontaneous tumors to guide its clinical development as a cancer therapeutic for human patients.
Liquid crystalline poly(1-alkynes) with different functional bridges and lengths of alkyl spacer {HCC(CH
2)
3OBiphOCO(CH
2)
10CH
3
n
—
1(3); Biph=4,4′-biphenyl and HCC(CH
2)
m
OCOBiphOCO(CH
...2)
10CH
3
n
—
2(
m);
m=3, 4} are synthesized in high yields (up to 84%) by WCl
6–Ph
4Sn catalyst in dioxane at 60 °C. Polymer
1(3) shows atypical SmA texture. Focal-conic textures of SmA phase are, however, observed in
2(
m). Whereas
1(3) emits weakly at 367 nm, intense blue emissions (λ
max∼350 nm) are observed in
2(
m), whose intensity increases with the spacer length.
Acute myeloid leukemia (AML) is a near incurable disease with 5-year survival rates of 10% in older patients. Poor tolerance to chemotherapy, chemo resistance and high rate of relapse warrants less ...toxic and more effective regimens in AML. AML leads to a block in normal myeloid differentiation, leading to an accumulation of undifferentiated blasts. Thus, therapies directed towards promoting these cells to differentiate re-establishes the ability of AML cells to undergo cell death, reducing the leukemic population. Such differentiation therapy with all-trans retinoic acid (ATRA) has been successful in acute promyelocytic leukemia (APL), but not in other AML subtypes resistant to ATRA, thus presenting a promising and unexploited avenue for targeted therapies. There is accumulating evidence that Protein Kinase C (PKC) activation induces myeloid differentiation. However, since most PKC activators are either tumor promoters or have inconclusive activity, their potential for use in AML has yet to be realized. OSU-2S is a non-immunosuppressive derivative of FTY720 (a sphingosine analogue with anti-tumorigenic properties) and has previously been reported to directly activate PKC to mediate cytotoxicity, showing promising in vitro and in vivo activity against a number of leukemias and lymphomas, and other malignancies such as hepatocellular carcinoma. This impelled us to evaluate the activity of OSU-2S in causing cell death and differentiation in AML. We found that OSU-2S resulted in increased activation of PKC in HL-60 cells. In addition to effecting significant dose-dependent cytotoxicity against AML cell lines HL-60, MV4-11 and MOLM-13 mean decrease in viability with OSU-2S in HL-60: 85±7% (p=0.08), MV4-11: 74±20% (p=0.02), MOLM-13: 60±25% (p=0.04), and in primary samples from leukemic patients, including high risk FLT3-ITD mutated AMLs (65±8% decrease in viability with OSU-2S, p<0.0001, n=13, dose trend p<0.0001), OSU-2S caused a significant upregulation of CD11b, a cell-surface marker associated with myeloid maturation, in HL-60 and primary AML samples (25±5% and 14±7% increase in CD11b+ cells with OSU-2S vs vehicle control in HL-60 (p =0.0126) and primary AML (p=0.043) cells respectively. In addition, OSU-2S treated HL-60 and primary AML cells exhibited morphological features indicative of differentiation, as evidenced by lower nuclear to cytoplasmic ratio, and highly vacuolated and granulated cytoplasm. The observed differentiation activity prompted us to test if OSU-2S affected the leukemia-initiating cell population. Colony forming assays showed OSU-2S significantly reduced the number of colony forming units-leukemic (CFU-L) in primary AML blasts (52±13% decrease in CFU-L in response to OSU-2S vs vehicle control; p=0.018, n=4). OSU-2S mediated cytotoxicity was significantly abrogated in the presence of PKC inhibitor Bisindolylmaleimide (BIS) in HL-60 cell line 39±8% decrease in OSU-2S induced cytotoxicity in the presence of BIS vs control (p=0.008), n=3 as well as in primary AML cells 41±16% decrease in OSU-2S induced cytotoxicity (p=0.02), n=6, indicating that OSU-2S mediates its cytotoxic effects in AML via PKC activation.
To further gain insight into the mechanism of cell death and differentiation effected by OSU-2S, we performed RNA-Sequencing analysis of total RNA isolated from MV4-11 cells treated with OSU-2S. Ingenuity Pathway Analysis on these transcripts showed significant changes in gene expression affecting cell cycle regulation and proliferation (p=10-13). Cell cycle analysis of MV4-11, HL-60 and U-937 cells by PI staining followed by flow cytometry confirmed a decrease in cells in G2/M phase of the cell cycle associated with increased cells in S but not Go/G1 phase (69.63% decrease in G2/M phase cells with OSU-2S vs vehicle control, p=0.04). Current ongoing studies in cell line-derived xenografted mouse models of AML indicate favorable therapeutic effect of OSU-2S in providing survival advantage in AML. Tumor burden monitoring by bioluminescent imaging shows reduction in tumor burden following OSU-2S treatment. Our results show promising pre-clinical efficacy of OSU-2S against AML, and warrants further studies to establish clinical efficacy as a potential therapeutic agent. Ongoing studies are also directed towards studying the involvement and role of PKC and other target molecules in the cell death and differentiation effected by OSU-2S.
Walker:Gilead: Research Funding. Mims:Novartis: Honoraria. Chen:The Ohio State University: Patents & Royalties: OSU-2S. Byrd:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding; Janssen: Research Funding; Genentech: Research Funding; The Ohio State University: Patents & Royalties: OSU-2S. Vasu:Boehringer-Ingelheim: Research Funding; Stemline Therapeutics: Research Funding.
A conjugated polymer consisting of carbazole and quinoxaline units (5) is synthesized in a high isolation yield (95%). The polymer possesses a high molecular weight (∼22 kDa) and is completely ...soluble in common solvents. When 5 is used as an emitting layer in a multilayer electroluminescence (EL) device, it emits a blue light of 484 nm and exhibits a current efficiency of ∼0.7 cd/A.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
TP53 encodes for tumor protein p53. The suppression of p53 protein results in interruption of DNA repair mechanisms in dividing malignant cells thereby increasing the DNA damage and activating ...p53-independent mechanisms of apoptosis. This ultimately may translate into enhanced cytotoxic effects of standard chemotherapy. Based on this rationale, Cenersen, a phosphorothioate oligonucleotide antisense to p53-mRNA was synthesized and tested in clinical trials for patients with acute myeloid leukemia (AML). An important component of Cenersen clinical development is to develop a sensitive and specific method to quantify plasma and intracellular levels of Cenersen in different biologic matrices in order to determine tissue and intracellular distribution of the parent compound and its metabolites. Ultimately, this will allow us to determine pharmacokinetic and pharmacodynamic relationship for dose-effect correlation and design effective regimen to be rapidly translate into the clinic. An ELISA-based assay was adapted for assay development and validation of Cenersen in mouse plasma and cell lysate. Cellular uptake of Cenersen was studied in MV4-11 and KASUMI-1 AML cell lines. Real-time RT-PCR was used to measure P53-mRNA expression changes in treated cells. The assay had a limit of quantification of 35pmol/L in mouse plasma. Within-day and between-day precision of <15% and accuracy nearly 100% were observed in a linear range of 10-2000pmol/L (R(2)=0.99) in AML cell lysate. The selectivity of this assay examined as cross-reactivity with its 3'N-1, 3'N-2-metabolites, was 16.8% and 0.4%, respectively, and with its mismatch and the scramble oligonucleotides was 0.06% and 0.4%, respectively. Cenersen was stable in mouse plasma up to 8h at 37°C. When exposed to 0.1-1μmol/L Cenersen, MV4-11 and KASUMI-1 cells showed intracellular concentration in the range of 9.97-45.34nmol/mg protein and 0.1-2.1nmol/mg protein, respectively. Successful downregulation of p53-mRNA expression was observed in Cenersen treated cells. This ELISA-based assay was applicable to plasma and intracellular concentration measurement of Cenersen. Assessment of achievable concentration of Cenersen in different biologic matrices will be useful to elucidate the biological and clinical activity of this promising drug and define its recommended dose in future clinical trials.
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