This fact allowed some structure/activity relationship studies that .. common ones belong to the indole and 2-phenylethylamine groups . Imatinib, the first clinically viable Abl kinase inhibitor, is the result of a medicinal chemistry project Analysis of structure activity relationships showed that. Jun 18, Though PGG was reported to have antitumor activities in breast, prostate, activity of imatinib and suppresses the growth of K cells in mice.
Therefore, we analyzed 13 CpG sites located in the CpG island of the promoter region from — bp to — bp upstream the transcription start Figure 5A.
MicroRNA-212/ABCG2-axis contributes to development of imatinib-resistance in leukemic cells
For ABCG2, we focused on four CpGs in the immediate proximity of the transcription start — to — bp upstream the transcription startFigure 5D as there is no CpG island located in the promoter region. Consistent with our results of the protein and gene expression analysis Supplementary Figure 1we did not observe significant alterations in methylation of the ABCB1 promotor during the development of IM-resistance Supplementary Figure 4.
A Graphical overview of the miR promoter. Methylation of 13 CpGs within region — to — bp upstream of the first nucleotide of the miR gene was analyzed using bisulfite-pyrosequencing. Every line indicates one CpG. In this study, we analyzed whether alteration of the endogenous miR level led to changes in IM-susceptibility in the K CML cell line.
We showed that during the development of IM-resistance, miR expression was downregulated at low IM-concentrations 0. In fact, we could mimic this mechanism by miR inhibition and blockade of miR function in IM-sensitive cells.
In this context, the inhibition of miR followed by upregulation of ABCG2 might lead to reduction of intracellular IM-concentrations. Our data suggest that these mechanisms contribute to acute resistance against low doses of IM. However, the role of these two transporters in IM-resistance is controversially discussed. There are also controversial findings on the role of the uptake transporter organic cation transporter 1 OCT1 [ 3132 ], suggesting however that for K cells, OCT1 is of minor if any importance.
In contrast, the IM-concentrations we used in our experiments ranged between 0. Moreover, due to several reasons, not all CML cells will be exposed to the full dose. Second, imatinib concentration varies over time, being influenced by genetic factors, esp. CYP3A4 inhibitors and inducers [ 35 — 38 ]. Our data suggest that exposure to low concentrations of imatinib could contribute to the phenomena of imatinib-resistance, hence our data is important for the understanding of the molecular processes.
ABCG2 itself is highly expressed in stem cells and it was shown that changes in ABCG2 protein expression influence cell survival during the development of IM-resistance [ 4111339 ]. Our findings are in line with the concept of a variable, dynamic expression of drug transporters during the development of resistance [ 12 ].
At higher IM doses, beyond 0. It seems likely that other mechanisms of resistance such as upregulation of other transporters [ 40 — 42 ] or drifts to alternate pathways take place [ 43 ]. Furthermore, we investigated if methylation was affected during the development of IM-resistance. It is widely known that global changes in methylation occur in cancer, often leading to hypermethylation on tumor suppressor genes and hypomethylation of oncogenes [ 44 ].
We observed a hypermethylation of miR promoter as well as an elevated miR expression in high dose-IM-resistant cells, compared to 0. Nevertheless, it was shown that miR promoter hypermethylation resulted in low miR expression and pronounced tumor metastasis and poor prognosis in gastric cancer [ 3047 ].
So, we suppose that hypermethylation of the examined CpGs results in less binding of these transcriptional repressors and subsequently, pronounced transcription of miR [ 4849 ]. Still, it is not clear if the observed hypermethylation is a cause or consequence of IM-treatment. Further experiments could now reveal how miR hypermethylation is regulated and might contribute to IM-resistance.
Nevertheless, our results point to a potential epigenetic regulation of miR expression during the development of IM-resistance. We did not observe any changes in methylation of these 4 CpGs during the development of IM-resistance, these findings are in line with those of different cancer entities, such as glioblastoma multiforme [ 50 ], breast-cancer [ 26 ] or multiple myeloma [ 51 ], where no or only partial impact of ABCG2 methylation could be observed.
This is in line with the absence of ABCB1 in the tested cell lines. Nevertheless, it is well known that a single microRNA is able to bind and regulate multiple targets in parallel [ 52 ]. MiR expression is altered in a variety of tumors such as non-small cell lung cancer or hepatocellular carcinoma. In hematologic malignancies, especially chronic lymphocytic leukemia, miR is dysregulated in B cell development and proliferation, being upregulated by B cell receptor stimulation.
Consequently, analysis of other pathways and miR targets could reveal further mechanisms causing the observed miR effect on IM-susceptibility, especially in cells that underwent long-term or high dose treatment with IM.
We tested several pre-miR-concentrations, lying within the non-toxic range with lowest off target-effect [ 57 ], but did not observe any effects after transfection. In IM-resistant cells, however, additional transfection of miRmimic or -inhibitor did not result in significant changes of cell viability or apoptosis as well. In these cells, other mechanisms could protect the cell from miRmediated effects on ABCG2 expression, such as intracellular depletion of miR or activation of counteracting pathways.
Interestingly, the effects of miR inhibition on apoptosis and cytotoxicity could not be significantly increased by dose doubling of the inhibitor. Potentially, the endogenous amount of miR was already blocked by the lower dose of miR inhibitor.
Therefore, we used the lower inhibitor concentration for further experiments.Structure Activity Relationship of Benzodiazepines (BZDs)
It is well known that the variable activity or expression of drug transporters leads to differences in drug efflux and turnover [ 5859 ]. These findings could reveal new insights into the initiation of IM-resistance. IM-resistant cells were generated as previously described [ 116162 ]. Cells were considered to be resistant, when the proliferation rate under the respective IM treatment was restored. During the course of the IM-resistance, cells resistant to distinct concentrations, referred to as K cells 0.
Sequencing reaction was performed using the previously described primers at the institute of clinical molecular biology, UKSH, Kiel [ 6364 ]. The used concentrations were proven as non-toxic for the cells. Analysis of effects on cell viability, apoptosis and cytotoxicity of pre-miR- or anti-miR-transfection on K cells 6 h after transfection, cells were subjected to various experiments to analyze the microRNA mimic or microRNA inhibitor effects on cell viability, apoptosis and survival.
After incubation, multiple assays were performed in parallel. In brief, a 1: The luminescence-based Caspase Glo 9 Assay Promega was used to investigate potential effects on apoptosis.
For analysis of cell survival, in terms of dead-cell protease activity released from membrane-disrupted cells, CytoTox-Glo Cytotoxicity Assay Promega was performed by using a 1: All assays were analyzed normalizing IM-treated to non-treated cells with subsequent statistical analyses as described in the statistic section. After three washing steps with TBST, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibody for 30 min mouse IgG NXA, 1: For further analysis, FlowJo v.
As positive control, K cells resistant to 0. For all sublines, 0. The supernatants were transferred into black, clear bottom 96 well-plates and detection was performed at the Infinite M pro device Tecan at nm for excitation and nm for emission.
Software and statistics DNA sequences hsa-miR Imatinib was a kind gift of Novartis GmbH Germany. We acknowledge financial support by the State of Schleswig-Holstein within the funding program Open Access Publikationsfonds. Bixby D, Talpaz M. Seeking the causes and solutions to imatinib-resistance in chronic myeloid leukemia. Arch Pathol Lab Med. Active transport of imatinib into and out of cells: The role of ABC transporters in clinical practice.
MicroRNA profiling in K cells under imatinib treatment: Development of imatinib and dasatinib resistance: Br J Clin Pharmacol. MiR modulates multidrug resistance via downregulation of ABCB1 in hepatocellular carcinoma cells. Band position and We analyzed the activated apoptotic pathways by performing absorption ranges are given in cm Proton nuclear magnetic Western blot analyses of caspase-8 and -9 proforms. Upon drug resonance 1H NMR spectra were recorded on Bruker and treatment caspase-8 and -9 became activated in most apoptotic MHz FT spectrometers in the indicated solvent.
Column cytochrome c release-dependent caspase-9 and -3 pathways. Aluminum oxide TLC cards from Fluka aluminum the caspase cascade. After 24 h, 14 significantly reduced the oxide precoated aluminum cards with fluorescent indicator at procaspase-8 immunoreactive band without changing the pro- nm and silica gel TLC cards from Fluka silica gel precoated caspase-9 intensity. On the other hand, lysates from 5-treated aluminum cards with fluorescent indicator at nm were used K cells showed no evidence of alteration in procaspase-8 for thin-layer chromatography TLC.
Elemental analysis results concentration and paralleled a dramatic decrease of procaspase-9 were found within 0. Ethyl ,Dihydrobenzoylpyrrolo[1,2-b]- 1 Faderl, S. The biology of chronic myeloid leukemia. New mixture of 14 1. Chronic 50 mL was refluxed overnight.
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After cooling, the mixture was myelogenous leukemia: The residue was purified by IntegratiVe Signaling through Abl: A Tyrosine Hz, 3H4. The leukaemic oncoproteins General Procedure for the Prepapartion of Derivatives Oncogenethiadiazepineacetate 5,5-Dioxide 9.
A mixture of 15 1. Oncogene19, After concentration the mixture was extracted with ethyl activation.
The survival function of the Bcr-Abl oncogene is mediated by column choromatography silica gel, dichloromethane. Blood86, A mixture of 19 9. Bcr-Abl-mediated resistance to apoptosis 0. The development of 3. Blood, After cooling, the mixture was poured onto crushed ice and 7 de Bree, F. Imitanib extracted with ethyl acetate, washed with brine, and dried.
Drugs Future26, Bloodpineacetate 5,5-Dioxide Yield 9 Soverini, S. ABL mutations in late chronic ,Dihydropyrrolo[1,2-b][1,2,5]benzothiadiazepine phase chronic myeloid leukemia patients with up-front cytogenetic carboxylic Acid 5,5-Dioxide Lithium hydroxide monohydrate resistance to imatinib are associated with a greater likelihood of 0. After dilution with water, Oncol. Synthesis of DNA-interactive pyrrolo- [2,1-c][1,4]benzodiazepines.
The acid was extracted with ethyl acetate, washed with brine, and 11 Tada-Oikawa, S.
Removal of the solvent gave pure To anhydrous aluminum 13 Artico, M. Then a solution of 16 0.
Research on compounds with antiblastic activity. Anthra- The organic layer was separated, washed with brine, and dried. Synthesis of pyrrolo[1,2-b][1,2,5]- Removal of the solvent gave crude 19, which was passed through benzothiadiazepine derivatives.