Biology subjects

Cinatl, J.

Publications and source records attributed to Cinatl, J..

7 recordsLinked to original sources

bioRxiv2018

Elevated expression of LGR5 and WNT signalling factors in neuroblastoma cells with acquired drug resistance

Neuroblastoma (NB) is the most common paediatric solid cancer with high fatality, relapses and acquired resistance to drug therapy. The clinical challenge NB poses requires new therapeutic approaches to improve survival rates.\n\nThe WNT signalling pathway is crucial in embryonic development but has also been reported to be dysregulated in glioblastoma, ovarian, breast and colorectal cancer. LGR5 is a receptor which potentiates the WNT/{beta}-catenin signalling pathway, hence contributing to cancer stem cell proliferation and self-renewal. LGR5 has been reported to promote both development and survival of colorectal cancer and glioblastomas.\n\nOur previous study illustrated that LGR5 is associated with aggressiveness in NB cell lines established at different stages of treatment. Following these findings, we investigated whether LGR5 is involved in acquired drug resistance via the WNT pathway in NB cell lines.\n\nCell lines in this study have an acquired drug resistance to vincristine (VCR) or doxorubicin (DOX).\n\nIn this study, we showed LGR5-LRP6 cooperation with enhanced expression of both proteins in SHSY5YrVCR, IMR32rDOX, IMR5rVCR and IMR5rDOX NB cell lines compared to paired parental cells. We also found elevated expression of {beta}-catenin in cell lines with acquired drug resistance is indicative of {beta}-catenin-dependent WNT signalling.\n\nThis study warrants further investigation into the role of the WNT signalling pathway in acquired drug resistance.

cancer biology↗

bioRxiv2018

TP53 mutations and drug sensitivity in acute myeloid leukaemia cells with acquired MDM2 inhibitor resistance

BackgroundMDM2 inhibitors are under investigation for the treatment of acute myeloid leukaemia (AML) patients in phase III clinical trials. To study resistance formation to MDM2 inhibitors in AML cells, we here established 45 sub-lines of the AML TP53 wild-type cell lines MV4-11 (15 sub-lines), OCI-AML-2 (10 sub-lines), OCI-AML-3 (12 sub-lines), and SIG-M5 (8 sub-lines) with resistance to the MDM2 inhibitor nutlin-3.\n\nMethods: Nutlin-3-resistant sub-lines were established by continuous exposure to stepwise increasing drug concentrations. The TP53 status was determined by next generation sequencing, cell viability was measured by MTT assay, and p53 was depleted using lentiviral vectors encoding shRNA.\n\nResultsAll MV4-11 sub-lines harboured the same R248W mutation and all OCI-AML-2 sub-lines the same Y220C mutation, indicating the selection of pre-existing TP53-mutant subpopulations. In concordance, rare alleles harbouring the respective mutations could be detected in the parental MV4-11 and OCI-AML-2 cell lines. The OCI-AML-3 and SIG-M5 sub-lines were characterised by varying TP53 mutations or wild type TP53, indicating the induction of de novo TP53 mutations. Doxorubicin, etoposide, gemcitabine, cytarabine, and fludarabine resistance profiles revealed a noticeable heterogeneity among the sub-lines even of the same parental cell lines. Loss-of-p53 function was not generally associated with decreased sensitivity to cytotoxic drugs.\n\nConclusionWe introduce a substantial set of models of acquired MDM2 inhibitor resistance in AML. MDM2 inhibitors select, in dependence on the nature of a given AML cell population, pre-existing TP53-mutant subpopulations or induce de novo TP53 mutations. Although loss-of-p53 function has been associated with chemoresistance in AML, nutlin-3-adapted sub-lines displayed in the majority of experiments similar or increased drug sensitivity compared to the respective parental cells. Hence, chemotherapy may remain an option for AML patients after MDM2 inhibitor therapy failure. Even sub-lines of the same parental cancer cell line displayed considerable heterogeneity in their response to other anti-cancer drugs, indicating the need for the detailed understanding and monitoring of the evolutionary processes in cancer cell populations in response to therapy as part of future individualised treatment protocols.

pharmacology and toxicology↗

bioRxiv2018

Incorporation of doxorubicin in different polymer nanoparticles and their anti-cancer activity

Nanoparticles are under investigation as carrier systems for anti-cancer drugs. They have been shown to accumulate in cancer tissues through the enhanced permeability and retention (EPR) effect, to reduce toxicity to non-target tissues, and to protect drugs from preliminary inactivation. However, nanoparticle preparations are not commonly compared for their anti-cancer effects at the cellular level. Here, we prepared doxorubicin-loaded nanoparticles based on poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), and PEGylated PLGA (PLGA-PEG) by solvent displacement and emulsion diffusion approaches. The resulting nanoparticles covered a size range between 73 and 246 nm. PLGA-PEG nanoparticle preparation by solvent displacement resulted in the smallest nanoparticles. In PLGA nanoparticles, the drug load could be optimised using solvent displacement at pH7 reaching 53 {micro}g doxorubicin/mg nanoparticle. In addition, these PLGA nanoparticles displayed sustained doxorubicin release kinetics compared to the more burst-like kinetics of the other preparations. In neuroblastoma cells, doxorubicin-loaded PLGA-PEG nanoparticles (presumably due to their small size) and PLGA nanoparticles prepared by solvent displacement at pH7 (presumably due to their high drug load and superior drug release kinetics) exerted the strongest anti-cancer effects. In conclusion, doxorubicin-loaded nanoparticles made by different methods from different materials displayed substantial discrepancies in their anti-cancer activity at the cellular level. Optimised preparation methods resulted in PLGA nanoparticles characterised by increased drug load, controlled drug release, and high anti-cancer efficacy. The design of drug-loaded nanoparticles with optimised anti-cancer activity at the cellular level is an important step in the development of improved nanoparticle preparations for anti-cancer therapy.

pharmacology and toxicology↗

bioRxiv2018

Drug repurposing: omeprazole increases the efficacy of acyclovir against herpes simplex virus type 1 and 2

ObjectivesOmeprazole was shown to improve the anti-cancer effect of the nucleoside-analogue 5-fluorouracil. Here, we investigated the effects of omeprazole on the activities of the antiviral nucleoside analogues ribavirin and acyclovir.\n\nMethodsWest Nile virus-infected Vero cells and influenza A H1N1-infected MDCK cells were treated with omeprazole and/ or ribavirin. Herpes simplex virus 1 (HSV-1)- or HSV-2-infected Vero or HaCat cells were treated with omeprazole and/ or acyclovir. Antiviral effects were determined by examination of cytopathogenic effects (CPE), immune staining, and virus yield assay. Cell viability was investigated by MTT assay.\n\nResultsOmeprazole concentrations up to 80g/mL did not affect the antiviral effects of ribavirin. In contrast, omeprazole increased the acyclovir-mediated effects on HSV-1- and HSV-2-induced CPE formation in a dose-dependent manner in Vero and HaCat cells. Addition of omeprazole 80g/mL resulted in a 10.8-fold reduction of the acyclovir concentration that reduces CPE formation by 50% (IC50) in HSV-1-infected Vero cells and in a 47.7-fold acyclovir IC50 reduction in HSV-1-infected HaCat cells. In HSV-2-infected cells, omeprazole reduced the acyclovir IC50 by 7.3-fold (Vero cells) or by 12.9-fold (HaCat cells). Omeprazole also enhanced the acyclovir-mediated effects on viral antigen expression and virus replication in HSV-1- and HSV-2-infected cells. In HSV-1-infected HaCat cells, omeprazole 80g/mL reduced the virus titre in the presence of acyclovir 1g/mL by 1.6x105-fold. In HSV-2-infected HaCat cells omeprazole 80g/mL reduced the virus titre in the presence of acyclovir 2g/mL by 9.2x103-fold. The investigated drug concentrations did not affect cell viability, neither alone nor in combination.\n\nConclusionsOmeprazole increases the anti-HSV activity of acyclovir. As clinically well-established and tolerated drug, it is a candidate drug for antiviral therapies in combination with acyclovir.

pharmacology and toxicology↗

bioRxiv2018

The structural basis for cancer drug interactions with the catalytic and allosteric sites of SAMHD1

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that depletes cellular dNTPs in non-cycling cells to promote genome stability and to inhibit retroviral and herpes viral replication. In addition to being substrates, cellular nucleotides also allosterically regulate SAMHD1 activity. Recently, it was shown that high expression levels of SAMHD1 are also correlated with significantly worse patient responses to nucleotide analogue drugs important for treating a variety of cancers, including Acute Myeloid Leukemia (AML). In this study, we used biochemical, structural, and cellular methods to examine the interactions of various cancer drugs with SAMHD1. We found that both the catalytic and the allosteric sites of SAMHD1 are sensitive to sugar modifications of the nucleotide analogs, with the allosteric site being significantly more restrictive. We crystallized cladribine-TP, clofarabine-TP, fludarabine-TP, vidarabine-TP, cytarabine-TP, and gemcitabine-TP in the catalytic pocket of SAMHD1. We find that all of these drugs are substrates of SAMHD1 and that the efficacy of most of these drugs is affected by SAMHD1 activity. Of the nucleotide analogues tested, only cladribine-TP with a deoxyribose sugar efficiently induced the catalytically active SAMHD1 tetramer. Together, these results establish a detailed framework for understanding the substrate specificity and allosteric activation of SAMHD1 with regards to nucleotide analogues, which can be used to improve current cancer and antiviral therapies.\n\nSignificanceNucleoside analogue drugs are widely used to treat a variety of cancers and viral infections. With an essential role in regulating the nucleotide pool in the cell by degrading cellular nucleotides, SAMHD1 has the potential to decrease the cellular concentration of frequently prescribed nucleotide analogues and thereby decrease their clinical efficacy in cancer therapy. To improve future nucleotide analogue treatments, it is important to understand SAMHD1 interactions with these drugs. Our work thoroughly examines the extent to which nucleotide analogues interact with the catalytic and allosteric sites of SAMHD1. This work contributes to the assessment of SAMHD1 as a potential therapeutic target for cancer therapy and the future design of SAMHD1 modulators that might improve the efficacy of existing therapies.

biochemistry↗

bioRxiv2017

Primary cilia mediate diverse kinase inhibitor resistance mechanisms in cancer

Primary cilia are microtubule-based organelles that detect mechanical and chemical stimuli. Although cilia house a number of oncogenic molecules (including Smoothened, KRAS, EGFR, and PDGFR), their precise role in cancer remains unclear. We have interrogated the role of cilia in acquired and de novo resistance to a variety of kinase inhibitors, and found that in several examples, resistant cells are distinctly characterized by an increase in the number and/or length of cilia with altered structural features. Changes in cilia length seem to be linked to the lack of recruitment of Kif7 and IFT81 to cilia tips, and result in enhanced hedgehog pathway activation. Notably, Kif7 knockdown is sufficient to confer drug resistance in drug sensitive cells. Conversely, targeting of cilia length or integrity through genetic and pharmacological approaches overcomes kinase inhibitor resistance. The identification of a broad mechanism of pathway-unbiased drug resistance, represents a major advancement in oncology, and helps define a specific and important role for cilia in human cancer.

cell biology↗

bioRxiv2017

A Cell Culture Platform For Cryptosporidium That Enables Long-Term Cultivation And New Tools For The Systematic Investigation Of Its Biology

Cryptosporidium parasites are a major cause of diarrhoea that pose a particular threat to children in developing areas and immunocompromised individuals. Curative therapies and vaccines are lacking. Currently, Cryptosporidium oocysts for research must be freshly produced in animals and cannot be long-term stored. Here, we show that COLO-680N cells infected with two different Cryptosporidium parvum strains (Moredun, Iowa) produce sufficient infectious oocysts to infect subsequent cultures. Oocyst identity was confirmed by specific staining (Crypt-a-glo, Vicia Villosa lectin, Sporo-glo), PCR-based amplification of Cryptosporidium-specific genes, lipidomics fingerprinting, and atomic force microscopy (AFM). Antibody-stained oocysts produced unstained oocysts confirming production of novel oocysts. Infected cultures could be cryoconserved and continued to produce infectious oocysts after resuscitation. Transmission electron microscopy identified all key Cryptosporidium life cycle stages. Infected cultures produced thick-walled (primarily involved in Cryptosporidium transmission between organisms) and thin-walled oocysts (important for Cryptosporidium propagation within a host/tissue) as indicated by DAPI staining (only thin-walled oocysts are permeable to DAPI staining, thus allowing visualisation of sporozoites) and AFM. In conclusion, we present a novel, easy-to-handle cell culture system that enables the propagation, cryopreservation and detailed investigation of Cryptosporidium at a laboratory scale. Its availability will accelerate research on Cryptosporidium and the development of anti-Cryptosporidium drugs.

microbiology↗