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Background: SAMHD1 mediates resistance to anti-cancer nucleoside analogues, including cytarabine, decitabine, and nelarabine that are commonly used for the treatment of leukaemia, through cleavage of their triphosphorylated forms. Hence, SAMHD1 inhibitors are promising candidates for the sensitisation of leukaemia cells to nucleoside analogue-based therapy. Here, we investigated the effects of the cytosine analogue CNDAC, which has been proposed to be a SAMHD1 inhibitor, in the context of SAMHD1. Methods: CNDAC was tested in 13 acute myeloid leukaemia (AML) cell lines, in 26 acute lymphoblastic leukaemia (ALL) cell lines, ten AML sublines adapted to various antileukaemic drugs, 24 single cell-derived clonal AML sublines, and primary leukaemic blasts from 24 AML patients. Moreover, 24 CNDAC-resistant sublines of the AML cell lines HL-60 and PL-21 were established. The SAMHD1 gene was disrupted using CRISPR/Cas9 and SAMHD1 depleted using RNAi, and the viral Vpx protein. Forced DCK expression was achieved by lentiviral transduction. SAMHD1 promoter methylation was determined by PCR after treatment of genomic DNA with the methylation-sensitive HpaII endonuclease. Nucleoside (analogue) triphosphate levels were determined by LC-MS/MS. CNDAC interaction with SAMHD1 was analysed by an enzymatic assay and by crystallisation. Results: Although the cytosine analogue CNDAC was anticipated to inhibit SAMHD1, SAMHD1 mediated intrinsic CNDAC resistance in leukaemia cells. Accordingly, SAMHD1 depletion increased CNDAC triphosphate (CNDAC-TP) levels and CNDAC toxicity. Enzymatic assays and crystallisation studies confirmed CNDAC-TP to be a SAMHD1 substrate. In 24 CNDAC-adapted acute myeloid leukaemia (AML) sublines, resistance was driven by DCK (catalyses initial nucleoside phosphorylation) loss. CNDAC-adapted sublines displayed cross-resistance only to other DCK substrates (e.g. cytarabine, decitabine). Cell lines adapted to drugs not affected by DCK or SAMHD1 remained CNDAC sensitive. In cytarabine-adapted AML cells, increased SAMHD1 and reduced DCK levels contributed to cytarabine and CNDAC resistance. Conclusion: Intrinsic and acquired resistance to CNDAC and related nucleoside analogues are driven by different mechanisms. The lack of cross-resistance between SAMHD1/ DCK substrates and non-substrates provides scope for next-line therapies after treatment failure.
Combination chemotherapy with gemcitabine and cisplatin in patients with metastatic urothelial cancer of the bladder frequently results in the development of acquired drug resistance. Availability of cell culture models with acquired resistance could help to identify candidate treatments for an efficient second-line therapy. Six cisplatin- and six gemcitabine-resistant cell lines were established. Cell viability assays were performed to evaluate the sensitivity to 16 different chemotherapeutic substances. The activity of the drug transporter ATP-binding cassette transporter, subfamily B, member 1 (ABCB1, a critical mediator of multidrug resistance in cancer) was evaluated using fluorescent ABCB1 substrates. For functional assessment, cells overexpressing ABCB1 were generated by transduction with a lentiviral vector encoding for ABCB1, while zosuquidar was used for selective inhibition. In this study, 8 of 12 gemcitabine- or cisplatin-resistant cell lines were cross-resistant to carboplatin, 5 to pemetrexed, 4 to methotrexate, 3 to oxaliplatin, 5-fluorouracil, and paclitaxel, and 2 to cabazitaxel, larotaxel, docetaxel, topotecan, doxorubicin, and mitomycin c, and 1 of 12 cell lines was cross-resistant to vinflunine and vinblastine. In one cell line with acquired resistance to gemcitabine (TCC-SUPrGEMCI20), cross-resistance seemed to be mediated by ABCB1 expression. Our model identified the vinca alkaloids vinblastine and vinflunine, in Europe an already approved second-line therapeutic for metastatic bladder cancer, as the most effective compounds in urothelial cancer cells with acquired resistance to gemcitabine or cisplatin. These results demonstrate that this in vitro model can reproduce clinically relevant results and may be suitable to identify novel substances for the treatment of metastatic bladder cancer.
Background: West Nile virus (WNV) infection can cause severe meningitis and encephalitis in humans. Apoptosis was recently shown to contribute to the pathogenesis of WNV encephalitis. Here, we used WNV-infected glioma cells to study WNV-replication and WNV-induced apoptosis in human brain-derived cells. Results: T98G cells are highly permissive for lytic WNV-infection as demonstrated by the production of infectious virus titre and the development of a characteristic cytopathic effect. WNV replication decreased cell viability and induced apoptosis as indicated by the activation of the effector caspase-3, the initiator caspases-8 and -9, poly(ADP-ribose)polymerase (PARP) cleavage and the release of cytochrome c from the mitochondria. Truncation of BID indicated cross-talk between the extrinsic and intrinsic apoptotic pathways. Inhibition of the caspases-8 or -9 inhibited PARP cleavage, demonstrating that both caspases are involved in WNV-induced apoptosis. Pancaspase inhibition prevented WNV-induced apoptosis without affecting virus replication. Conclusions: We found that WNV infection induces cell death in the brain-derived tumour cell line T98G by apoptosis under involvement of constituents of the extrinsic as well as the intrinsic apoptotic pathways. Our results illuminate the molecular mechanism of WNV-induced neural cell death.
Background Drug resistance to chemotherapy is often associated with increased malignancy in neuroblastoma (NB). One explanation for the link between resistance and malignancy might be that resistance facilitates cancer progression and invasion. To investigate this hypothesis, adhesion, transendothelial penetration and NCAM (CD56) adhesion receptor expression of drug-resistant versus drug-sensitive NB tumor cells were evaluated. Methods Acquired drug resistance was mimicked by exposing parental UKF-NB-2, UKF-NB-3 or IMR-32 tumor cells to increasing concentrations of vincristine- (VCR) or doxorubicin (DOX) to establish the resistant tumor cell sublines UKF-NB-2VCR, UKF-NB-2DOX, UKF-NB-3VCR, UKF-NB-3DOX, IMR-32VCR and IMR-32DOX. Additionally, the malignant behaviour of UKF-NB-4, which already possessed the intrinsic multidrug resistance (MDR) phenotype, was analyzed. UKF-NB-4 exposed to VCR or DOX were designated UKF-NB-4VCR or UKF-NB-4DOX. Combined phase contrast - reflection interference contrast microscopy was used to separately evaluate NB cell adhesion and penetration. NCAM was analyzed by flow cytometry, western blot and RT-PCR. Results VCR and DOX resistant tumor sublines showed enhanced adhesion and penetration capacity, compared to their drug naive controls. Strongest effects were seen with UKF-NB-2VCR, UKF-NB-3VCR and IMR-32DOX. DOX or VCR treatment also evoked increased invasive behaviour of UKF-NB-4. The process of accelerated tumor invasion was accompanied by decreased NCAM surface and protein expression, and down-regulation of NCAM coding mRNA. Transfection of UKF-NB-4VCR cells with NCAM cDNA led to a significant receptor up-regulation, paralleled by diminished adhesion to an endothelial cell monolayer. Conclusions It is concluded that NB cells resistant to anticancer drugs acquire increased invasive capacity relative to non-resistant parental cells, and that enhanced invasion is caused by strong down-regulation of NCAM adhesion receptors.
Mitte März 2003 löste die WHO einen weltweiten Alarm aus, nachdem sich eine neuartige, schwere und unter bestimmten Umständen hochansteckende Atemwegserkrankung scheinbar unaufhaltsam über weite Teile der Welt auszubreiten schien. Am 15. März desselben Jahres landeten die ersten Patienten mit Verdacht auf Schweres Akutes Respiratorisches Syndrom (SARS) in Frankfurt und wurden auf die Isolierstation des Universitätsklinikums aufgenommen. Auslöser war ein zuvor nicht bekanntes Coronavirus, das heute als SARS-CoV bezeichnet wird. Derzeit laufen Untersuchungen zur Biologie und Epidemiologie des neuen Erregers, zu antiviralen Hemmstoffen sowie zu Desinfektions- und Inaktivierungsmöglichkeiten und neuen Therapieoptionen. Daneben wird analysiert, wie sich das öffentliche Gesundheitswesen auf eine mögliche Wiederkehr vorbereiten muss. SARS ist ein Beispiel dafür, wie schnell sich eine Infektionskrankheit in der modernen Welt international ausbreiten kann und wie wichtig in einem solchen Falle eine gut koordinierte internationale Kooperation ist. Frankfurter Forscher berichten.
The antineoplastic alkaloid ellipticine is a prodrug, the pharmacological efficiency of which is dependent on its cytochrome P450 (CYP)- and/or peroxidase-mediated activation to species forming DNA adducts in target tissues. Here, we found that this compound is cytotoxic to human BHT-101, B-CPAP and 8505-C thyroid cancer cells and blocks one or more phases of cell cycle in these cancer cells. Ellipticine toxicity to the thyroid cancer cells corresponded to levels of DNA adducts generated by the CYP- and/or peroxidase-mediated ellipticine metabolites, 12-hydroxy- and 13-hydroxyellipticine, in these cells. Cultivation of all tested cells under hypoxic conditions (1 % oxygen) led to a decrease in ellipticine toxicity. Such a lower sensitivity of cells to ellipticine correlates with a decrease in the formation of ellipticine-derived DNA adducts in these cells. Using Western blotting, the expression of CYP1A1, 1B1, 3A4, thyroid peroxidase (TPO), cyclooxygenase-1 (COX-1) and cytochrome b5, the enzymes that catalyze, and/or influence ellipticine metabolism, was investigated in the cancer cells. Furthermore, the effects of ellipticine treatment on the expression levels of these proteins in thyroid cancer cells were also examined. The results indicate that the highest expression levels of cytochrome b5 together with CYP1A1 and 3A4 determine the highest DNA adduct formation and cytotoxicity of ellipticine in B-CPAP cells. They also demonstrate that formation of covalent DNA adducts by ellipticine is the predominant mechanism responsible for its cytotoxicity in studied cells.
The development of resistance to chemotherapeutic agents, such as Doxorubicin (DOX) and cytarabine (AraC), is one of the greatest challenges to the successful treatment of Acute Myeloid Leukemia (AML). Such acquisition is often underlined by a metabolic reprogramming that can provide a therapeutic opportunity, as it can lead to the emergence of vulnerabilities and dependencies to be exploited as targets against the resistant cells. In this regard, genome-scale metabolic models (GSMMs) have emerged as powerful tools to integrate multiple layers of data to build cancer-specific models and identify putative metabolic vulnerabilities. Here, we use genome-scale metabolic modelling to reconstruct a GSMM of the THP1 AML cell line and two derivative cell lines, one with acquired resistance to AraC and the second with acquired resistance to DOX. We also explore how, adding to the transcriptomic layer, the metabolomic layer enhances the selectivity of the resulting condition specific reconstructions. The resulting models enabled us to identify and experimentally validate that drug-resistant THP1 cells are sensitive to the FDA-approved antifolate methotrexate. Moreover, we discovered and validated that the resistant cell lines could be selectively targeted by inhibiting squalene synthase, providing a new and promising strategy to directly inhibit cholesterol synthesis in AML drug resistant cells.
Previous studies reported on the safety and applicability of mesenchymal stem/stromal cells (MSCs) to ameliorate pulmonary inflammation in acute respiratory distress syndrome (ARDS). Thus, multiple clinical trials assessing the potential of MSCs for COVID-19 treatment are underway. Yet, as SARS-inducing coronaviruses infect stem/progenitor cells, it is unclear whether MSCs could be infected by SARS-CoV-2 upon transplantation to COVID-19 patients. We found that MSCs from bone marrow, amniotic fluid, and adipose tissue carry angiotensin-converting enzyme 2 and transmembrane protease serine subtype 2 at low levels on the cell surface under steady-state and inflammatory conditions. We did not observe SARS-CoV-2 infection or replication in MSCs at steady state under inflammatory conditions, or in direct contact with SARS-CoV-2-infected Caco-2 cells. Further, indoleamine 2,3-dioxygenase 1 production in MSCs was not impaired in the presence of SARS-CoV-2. We show that MSCs are resistant to SARS-CoV-2 infection and retain their immunomodulation potential, supporting their potential applicability for COVID-19 treatment.
Adaptation of wild-type p53 expressing UKF-NB-3 cancer cells to the murine double minute 2 inhibitor nutlin-3 causes de novo p53 mutations at high frequency (13/20) and multi-drug resistance. Here, we show that the same cells respond very differently when adapted to RITA, a drug that, like nutlin-3, also disrupts the p53/Mdm2 interaction. All of the 11 UKF-NB-3 sub-lines adapted to RITA that we established retained functional wild-type p53 although RITA induced a substantial p53 response. Moreover, all RITA-adapted cell lines remained sensitive to nutlin-3, whereas only five out of 10 nutlin-3-adapted cell lines retained their sensitivity to RITA. In addition, repeated adaptation of the RITA-adapted sub-line UKF-NB-3rRITA10 μM to nutlin-3 resulted in p53 mutations. The RITA-adapted UKF-NB-3 sub-lines displayed no or less pronounced resistance to vincristine, cisplatin, and irradiation than nutlin-3-adapted UKF-NB-3 sub-lines. Furthermore, adaptation to RITA was associated with fewer changes at the expression level of antiapoptotic factors than observed with adaptation to nutlin-3. Transcriptomic analyses indicated the RITA-adapted sub-lines to be more similar at the gene expression level to the parental UKF-NB-3 cells than nutlin-3-adapted UKF-NB-3 sub-lines, which correlates with the observed chemotherapy and irradiation sensitivity phenotypes. In conclusion, RITA-adapted cells retain functional p53, remain sensitive to nutlin-3, and display a less pronounced resistance phenotype than nutlin-3-adapted cells.
The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is partly under control by vaccination. However, highly potent and safe antiviral drugs for SARS-CoV-2 are still needed to avoid development of severe COVID-19. We report the discovery of a small molecule, Z-Tyr-Ala-CHN2, which was identified in a cell-based antiviral screen. The molecule exerts sub-micromolar antiviral activity against SARS-CoV-2, SARS-CoV-1, and human coronavirus 229E. Time-of-addition studies reveal that Z-Tyr-Ala-CHN2 acts at the early phase of the infection cycle, which is in line with the observation that the molecule inhibits cathepsin L. This results in antiviral activity against SARS-CoV-2 in VeroE6, A549-hACE2, and HeLa-hACE2 cells, but not in Caco-2 cells or primary human nasal epithelial cells since the latter two cell types also permit entry via transmembrane protease serine subtype 2 (TMPRSS2). Given their cell-specific activity, cathepsin L inhibitors still need to prove their value in the clinic; nevertheless, the activity profile of Z-Tyr-Ala-CHN2 makes it an interesting tool compound for studying the biology of coronavirus entry and replication.