Monthly Archives: July 2019

The by-product of the methyl transfer reaction has been shown to inhibit MTase activities for WNV

DENV2 and DENV3. The IC50 values for inhibition of the WNV and DENV3 MTase activities by AdoHcy were estimated to be in low micromolar or even nanomolar range. In contrast, our results showed that AdoHcy and its derivatives do not significantly inhibit both the N-7 and 2′-O activities for MTases from four different viruses, even at very high concentrations. Although there are some differences in assay conditions such as buffers, pH, substrates, and constructs of enzymes used, it is hard to believe that they will account for the large discrepancies. As shown in Figure 3, in the absence of the positive control SIN inhibitor, all enzymes could efficiently carry out the N-7 and 2′-O MTase reactions in our experiments. Under the same conditions as for AdoHcy, SIN could efficiently abolish the MTase activities of all MTases from the four viruses, which is consistent with our previous results showing that SIN inhibited both MTase activities of the WNV MTase with IC50 about 14 ��M using the TLC method. In contrast, AdoHcy under the same conditions failed to inhibit the enzymatic activities. The discrepancies may more reasonably be attributed to the different methods used to monitor the reactions. We monitored the reaction product, m7G*pppA and double methylated m7G*pppAm, using the TLC method. Although this method is low throughput, its advantage is the ability to directly “visualize” and quantify the reaction product. Alternative higher throughput monitoring methods could possibly quantify non-specific binding of radiolabeled materials and/or signals arising from incorporation of radio-labeled materials to other positions of RNA. Previous studies employed the SPA-based scintillation assay in which -AdoMet was used as a co-factor and activity was monitored by scintillation counting of the transfer of -labeled methyl group to the viral RNA. Nonspecific binding of radio-labeled materials or incorporation of radio-labeled materials to positions other than N-7 and 2′-O of the RNA could affect the activity reported by this assay. It was reported that N-7 and 2′-O reactions might only account for one-third of the total signals and that a large fraction of signals were unresolved when using the SPA method. In particular, the flavivirus MTase was reported to also carry out 2′-O methylation of internal adenosines in the viral RNA. The unresolved signals therefore could be from methylations of internal adenosines of the RNA. The Fulvestrant Estrogen Receptor inhibitor presence of these unresolved signals may thus affect how the results from inhibition studies using the SPA method were interpreted. It is possible that AdoHcy might mainly inhibit the internal methylation activity of flavivirus MTase, for which the hypothesis requires further investigation. The weak inhibition of the N-7 and 2′-O activities of flavivirus by AdoHcy are consistent with functional analysis indicating that it does not suppress viral growth till a high concentration is reached. In contrast, SIN inhibits both N-7 and 2′-O activities of the WNV MTase with IC50 of 14 ��M in vitro, and can also efficiently inhibit the growth of WNV with an EC50 of 27 ��M. The ineffectiveness of AdoHcy in virus growth inhibition is also consistent with results from a number of studies showing that the circulating blood levels of AdoHcy are as high as 0.77 ��M, and the levels of AdoMet are as high as 2.6 ��M. The binding affinity of AdoHcy for the DENV3 MTase was also shown to be much lower than those of AdoMet and SIN. The low affinity of AdoHcy for the MTase may facilitate the by-product release from the MTase and replenishment with a fresh AdoMet for a new cycle of methylation reaction. Structural comparison also supports the results. Superposition of the crystal structures of the WNV MTase-SIN and MTase-AdoHcy complexes reveals that SIN binds to the AdoMet pocket of the MTase in a conformation similar to that of AdoHcy in the MTase-AdoHcy complex. However, the free amine NE of the C-NH2 group of SIN, i.e., the group that replaces the SCH3 group of AdoMet, makes at least five additional contacts with the MTase, which include a pair of potential hydrogen bonds between the NE atom of SIN and the OD1 and O atoms of the MTase catalytically Reversine essential residue D146. The structural results correlate very well with MM-PBSA analysis of binding of SIN and AdoHcy to the WNV MTase, which showed that SIN binds the WNV MTase more favorably than AdoHcy by 6.8 kcal/mol, and that the NH2 group of SIN alone makes the largest contribution.

The hypermethylation of CGIs located in promoter regions of tumour suppressor genes is now recognized as an important mechanism for gene inactivation

AZA and DAC treated samples were not associated with CGIs, and no array based methylation data was obtained for these genes. However, DNA demethylation was detected in the non-CGI promoters of the top three up-regulated genes by bisulfite sequencing. These data indicate that prolonged low dose treatments are capable of demethylating CpG sites at non-CGI promoters and that this may have an effect on gene expression. The impact of demethylating agents on AML cell lines has recently been evaluated in several studies using bisulfitemodified target DNA arrays. Here we have extended previous observations by investigating the effect of prolonged low-dosage treatment with AZA and DAC in a model, which is likely to be more similar to the clinical situation than previous short-term and/or high-dose treatments. Furthermore, we have investigated the effects in the SKM-1 cell line, which was derived from overt leukaemia following MDS and hence may provide a better model for investigating the relationship between demethylating treatments and MDS. We have used McrBC fragmentation in combination with standard CpG island arrays to robustly distinguish differential CGI methylation profiles in cells proliferating normally. Most of the CGIs are located at either TSS or within gene bodies. Gene-body CGIs are significantly more highly methylated than TSS CGIs. However, this epigenetic mark was preferentially lost at TSS CGIs after prolonged treatment with AZA or DAC. Demethylating agents are thought to act as nucleoside Niraparib side effects analogues that incorporate into DNA, causing specific inactivation of DNMT1. This effect is non-specific and cannot per se explain the selectivity of demethylation observed. In contrast, the de novo methyltransferase DNMT3B are targeted to specific loci and it is possible that their activity contributes to the specificity of the demethylation observed. However, we found a decrease in both DNMT1 and DNMT3B protein levels as a result of AZA or DAC treatment and hence it is unlikely that DNMT3B plays a strong role in the maintenance of DNA methylation at demethylation resistant loci. DNMT1 recognizes hemi-methylated DNA and causes the methylation of the non-methylated strand. A reduction in the level of active DNMT1 should thus lead to the presence of more hemi-methylated DNA VE-822 resulting in a passive demethylation during cell proliferation. It is not known whether DNMT1 is differentially targeted to different genomic regions in SKM-1 cells, but it is feasible that since DNMT1 recognizes hemi-methylated DNA, that it may be preferentially associated with regions of DNA containing high levels of methylated CpGs. In fact, genome-wide mapping data of DNMT family proteins suggests that DNMT1 is depleted in TSS and enriched in the gene bodies. On the other hand, active DNA demethylation mediated by the TET family of methylcytosine deoxygenases may also play a part in selectivity of demethylation. The Tet1 protein binds preferentially to TSSs and less intensively throughout gene bodies. Therefore, a reduction in overall activity of DNMTs may have a stronger demethylation effect at regions that are normally less methylated, such as promoter regions. A similar study on the effect of nanomolar-scale demethylating agents on both AML and breast cancer cell lines has recently been reported. The authors of this study concluded that low-dose DAC affected a sub-population of clonogenic cells, rather than directly inducing cytotoxicity, to produce an antitumor ��memory�� response. These effects were accompanied by sustained promoter demethylation and gene re-expression in key cellular regulatory pathways. In agreement with their data, genes involved in the immune response of the ‘Triggering Receptor Expressed on Myeloid Cells’ -1 signalling pathway were activated after low-dose DNMT inhibitor treatment. Moreover, we demonstrate that the enhanced expression of a subset of these molecules follows DNA demethylation during the course of treatment. Activation of the TREM-1 signalling pathway is a feature of mature differentiated myelomonocytic cells. TYROBP constitutively associates with TREM-1 to mediate the induction of intracellular signals that lead to inflammatory cytokine TNF-a and chemokine IL-8 production. Further investigation into the epigenetic regulation of the TREM-1 pathway may extend our knowledge of the molecular basis of hematopoiesis and myeloid cell differentiation.

Multiple biological effects related to acetylation of histone and non-histone proteins

The Compound Library chaperone heat shock protein 90. Vorinostat induces HSP90 hyperacetylation and inhibits its chaperone function. Thus, vorinostat may inhibit the growth of BCR-ABL-positive cells by changing BCR-ABL conformation via acetylation and inhibition of the chaperone protein HSP90. Phosphorylated cH2A.X is associated with early DNA damage and repair processes that occur in response to double-strand breaks in eukaryotic cells. Vorinostat induced growth arrest and apoptosis, thus aggravating the apoptotic and cytotoxic effects of ponatinib on Ba/F3 T315I mutant cells. Since imatinib inhibits STAT5 phosphorylation as well as the expression of STAT5 target genes, ponatinib may exhibit the same inhibitory effect. In our immunoblot assay, cH2A.X phosphorylation was detected after co-treatment with ponatinib and vorinostat. Co-treatment with ponatinib and vorinostat resulted in increased cytotoxicity and provided strong evidence that vorinostat augments ponatinibinduced apoptosis by enhancing DNA damage responses in BCRABL-positive cells. Patients with hematological malignancies, including Ph-positive leukemia, often AB1010 develop resistance to TKIs. In our study, we used Ba/F3 AP-R BCR-ABL cells and primary samples. We demonstrated that co-treatment with ponatinib and vorinostat reduced the proliferation of ponatinib-resistant cells. Therefore, ponatinib and vorinostat may affect the activity of BCR-ABL and increase antileukemic activity against BCR-ABL mutant cells. Recently, the use of ponatinib has been evaluated in other hematological malignancies and its use has been approved by the FDA. We previously isolated primary cells highly resistant to ponatinib showing several BCR-ABL point mutations. Thus, ponatinib resistance seems to be a possible concern in near future, and therefore, methods to overcome ABL TKI resistance need to be developed. In summary, our results provide new information on the molecular events underlying the antitumor activity of ponatinib and the HDAC inhibitor vorinostat. Co-treatment using these compounds together with molecular-targeted drugs will benefit those with BCR-ABL leukemic cells that are resistant to conventional treatments. Breast cancer is the second-leading cause of cancer death and morbidity among women worldwide. Gene expression profiling has revealed that breast cancer is a heterogeneous entity, and four primary molecular subgroups have been proposed: basal-like, luminal A, luminal B and human epidermal growth factor receptor 2 -overexpressed. Estrogen receptor /HER2positivebreast cancer belongs to the luminal B subtype and accounts for 20�C25% of all breast cancer cases. Studies have shown that ER+/HER2+ patients have a poor prognosis. In contrast to ER-positive/HER2-negtive breast cancer, ER+/HER2+ patients are less responsive to selective estrogen receptor modulators, such as tamoxifen, and to aromatase inhibitors. Recent studies have demonstrated that bidirectional crosstalk between ER and HER2 leads to endocrine resistance in ER+/ HER2+ breast cancer. Agents that block HER2, such as trastuzumaband lapatinib,improve the inhibitory effects of SERMs in ER+/HER2+ cancer. However, nearly 50% of ER+/HER2+ patients show no response. Furthermore, primary or acquired resistance to trastuzumab has been recognized as a major obstacle in the treatment of this disease. Several clinical trials have shown that combining HER2 inhibitors with SERMs improved progression-free survival but did not extend overall survival. Consequently, there is a significant need for elucidating the molecular signaling pathways that promote ER+/HER2+ breast cancer to enable the development of novel therapeutics. Interfering with the growth factor-driven signaling pathways and downstream effector.

Various cell lines with double mutations of K-Ras/BRAF and PIK3CA were resistant to AKT inhibitors

FACS analysis in a panel of endometrial cancer cell lines revealed a clear dose-dependent effect of NVP-BEZ235 on cell proliferation. NVPBEZ235 induces G1 arrest much more efficiently at a higher concentrationthan at a lower concentration. In contrast, RAD001 does not show evidence of such dose dependency. Previous reports also suggested that NVP-BEZ235 was more effective than rapalogs at higher concentrations. PI3K activity might not be sufficiently suppressed by 100 nM NVP-BEZ235, as indicated by the observation that decreased phosphorylation of Aktis not observed at 50 nM but is observed at 250 nM or higher. In addition, IC50 values were under 100 nM in cells from groups A and B. These data are in agreement with previous reports on other cancers that indicate a discrepancy between the basal activity of the PI3K/Akt pathway and the biochemical activity of NVP-BEZ235. Nevertheless, the dose-dependent antiproliferative activity at concentrations $250 nM suggests that the effect of NVP-BEZ235 was, at least in part, caused by inhibition of the PI3K/Akt pathway. Our data suggest that a dual inhibitor of PI3K/mTOR might be a more promising therapeutic strategy than a single mTOR inhibitor in endometrial cancer. Our in vivo studies in 2 cell lines of xenograft mice support the in vitro findings that inhibition of the PI3K/mTOR axis has an antitumor effect in endometrial cancers. We did not see any superior efficacy of NVP-BEZ235 in the in vivo study. The concentrations we used were 40 mg/kg for NVP-BEZ235 and 5 mg/kg for RAD001, which are equivalent with the previous invivo experiments. In a pharmacodynamic analysis, the levels of p-Akt, p-GSK3beta, p-FOXO1/3a, and p-S6 in tumors returned to the baseline levels within 24 h after administration of NVP-BEZ235, suggesting that inhibition of PI3K signaling by NVP-BEZ235 might not be sufficiently maintained over time. This is compatible with previous data showing that inhibition of p-Aktwas maintained for 16 h, with recovery to baseline levels at 24 h. It remains to be determined which oral dosing schedule is optimal in treatment of endometrial cancer. As well, the mechanisms of in-vivo antitumor effect by these drugs should be more clarified, as inhibition of mTOR might result in anti-angiogenic effect by suppressing HIF1-VEGF pathway. Developing predictive biomarkers in therapeutics targeting the PI3K/mTOR pathway is crucial, as alterations in several molecules are involved in the activation of this pathway. PIK3CA mutation and HER2 amplification have been recommended as useful biomarkers in breast cancer. Mutant oncogenic Ras has been suggested as a dominant determinant of resistance in several solid tumor cells. PTEN deficiency is controversial as a predictive biomarker. The mechanism of resistance in PTEN-deficient tumors might be explained by the predominant activation of p110beta in PTEN mutant tumors, as NVPBEZ235 and most of the other PI3K inhibitors suppress p110beta less preferentially than the other p110 isoforms. However, p110beta is not a predominant isoform in endometrial carcinomas with PTEN mutations. The significance of p110alpha in PTEN mutant endometrial cancer would be helpful to identify patients susceptible to NVP-BEZ235. Thus, the existence of PTEN mutations might be a predictive biomarker for the PI3K/mTOR inhibitors in endometrial carcinomas. Further in vivo analysis, including the anti-tumor effect of NVP-BEA235, RAD001, or a combination of these compounds with a MEK inhibitor on groups C and D tumors would be necessary to evaluate the utility of these factors as biomarkers. She et al reported and suggested that a MEK inhibitor sensitizes these double mutant cells to AKT or PI3K inhibitors.

Pathological reduction of ASM activity may be caused by mutations in the ASM gene itself

Recently, Lee et al.designed chemically modified peptidic derivates of a Taspase1 cleavage substrate. Although some of these compounds displayed mild SCH772984 inhibitory activity using in vitro Taspase1 assays, these peptide-based inhibitors have not shown efficacy in living cells, in contrast to our low molecular weight inhibitors. Although natural products appear to interrogate a different area of chemical space than synthetic compounds, the tested lipophilic fungal extracts showed no inhibitory activity. Failure may be due to the fact that albeit such extracts contain a mixture of many different substances, the concentration of potentially active ingredients may be too low or outweighed by toxic effects of other components. Also, the numbers of samples which have to be screened in unfocussed natural product libraries are usually high, and hit rates are mostly below 0.01%. Hence, as future strategies to identify potent Taspase1 inhibitors we suggest to focus on a rational synthesis of derivates based on the structures of our primary hits combined with HTS of large natural/synthetic compound libraries. Automatic thresholding using the Isodata algorithm was used to convert the image to a binary mask that included all fluorescence data above background. The Hoechst 33342 stainingmask was used to define the nuclear ROI. Subsequently, the Hoechst 33342 mask was subtracted from the GFP maskto create a staining mask defining the cytoplasmic ROI. Scans were performed sequentially with settings to give sub-saturating fluorescence intensity, and a minimum of 400 valid objects per well was recorded. Acid sphingomyelinaseis a lysosomal glycoprotein that catalyses the hydrolysis of sphingomyelin into ceramide and phosphorylcholine. Fusion of secretory lysosomes with the cell surface and translocation of lysosomal ASM onto the outer leaflet of the cell membrane plays an important role during stress response. CD95 ligands and cytokines such as tumor necrosis factor-a, interleukin-1 and interferon-c but also other stimuli including oxidative stress, reactive oxygen and nitrogen species, ionizing radiation, UV-C radiation, heat shock and other agents of stress, injury or infections by HIV or bacteria have been shown to stimulate ceramide production, assumed to be in part due to increased ASM activity. Ceramide, in turn, leads to membrane reorganization and downstream signalling that results in cell activation, very often cell stress or apoptosis. In addition to ASM, at least three other sphingomyelinases have been described in mammalian cells that vary in their pH optimum and cofactor dependency. Although these enzymes and an existing de novo synthesis pathway are alternative mechanisms for ceramide generation, activation of ASM itself has been proven to be critical for some cellular responses, such as WZ8040 apoptosis induced by reactive oxygen and nitrogen species, chemotherapy drugs such as cisplatin, bacteria, radiationand CD95. Furthermore, in contrast to other sphingomyelinases, ASM activity is tightly regulated. Ceramide is further metabolized to sphingosine and sphingosine-1-phosphate by acid ceramidaseand sphingosine kinases. While the biological function of sphingosine is largely unknown, sphingosine-1-phosphate has been shown to be involved in cellular differentiation, proliferation and cell migration. This dynamic balance between ceramide and sphingosine-1-phosphate is referred to as the “ceramide/sphingosine-1phosphate rheostat”, maintaining the balance between growth and cell death. ASM is best known for its involvement in Niemann-Pick disease, a lysosomal storage disease due to an inherited enzyme deficiency.