Robert O'Connor

Characterisation and manipulation of docetaxel resistant prostate cancer cell lines

BackgroundThere is no effective treatment strategy for advanced castration-resistant prostate cancer. Although Docetaxel (Taxotere®) represents the most active chemotherapeutic agent it only gives a modest survival advantage with most patients eventually progressing because of inherent or acquired drug resistance. The aims of this study were to further investigate the mechanisms of resistance to Docetaxel. Three Docetaxel resistant sub-lines were generated and confirmed to be resistant to the apoptotic and anti-proliferative effects of increasing concentrations of Docetaxel.ResultsThe resistant DU-145 R and 22RV1 R had expression of P-glycoprotein and its inhibition with Elacridar partially and totally reversed the resistant phenotype in the two cell lines respectively, which was not seen in the PC-3 resistant sublines. Resistance was also not mediated in the PC-3 cells by cellular senescence or autophagy but multiple changes in pro- and anti-apoptotic genes and proteins were demonstrated. Even though there were lower basal levels of NF-κB activity in the PC-3 D12 cells compared to the Parental PC-3, docetaxel induced higher NF-κB activity and IκB phosphorylation at 3 and 6 hours with only minor changes in the DU-145 cells. Inhibition of NF-κB with the BAY 11-7082 inhibitor reversed the resistance to Docetaxel.ConclusionThis study confirms that multiple mechanisms contribute to Docetaxel resistance and the central transcription factor NF-κB plays an immensely important role in determining docetaxel-resistance which may represent an appropriate therapeutic target.

Stable Aqueous Dispersions of Glycopeptide‐Grafted Selectably Functionalized Magnetic Nanoparticles

Bionanomaterials have received increasing attention over the last decade, owing to their potential to advance medical science by providing novel solutions for disease diagnostics and treatment. However, bionanomaterial engineering imposes considerable challenges, as significant design criteria and properties, which are dictated by the specific application, must be met. MRI-trackable magnetic nanoparticles (MNPs) and their assemblies, which are capable of targeting specific cell types, such as cancer cells, for image-guided treatment are increasingly being investigated. The typical design criteria for MNPs are biomedical in nature, including cellular selectivity (biorecognition), extended blood circulation times and biocompatibility, there are also practical considerations such as processability and scalability. Additional MRI-specific criteria include colloidal stability and high image contrast. The efficacy of an MNP suspension for generating contrast by enhancing, or suppressing, a local MRI signal is quantified by the spin–lattice (r1) and spin–spin (r2) relaxivities, respectively. These are the water relaxation enhancement per millimolar concentration of iron. For T1weighting applications (localized signal enhancement), r1 must be high, and r2 and the r2/r1 ratio must be low. Particle aggregation increases the r2 value, so T1-weighting can only be achieved by ensuring full MNP dispersion and long-term colloidal stability. Monosaccharides offer colloidal stability through charge and steric interactions along with the possibility of cellular targeting, and so have been used to stabilize MNPs. Recently, MNP clusters have been prepared by encapsulation of alkylstabilized MNPs using poly(maleic anhydride-alt-1-octadecene) followed by EDC coupling of aminated sugars. Detailed analysis of the effect of coating on cellular uptake showed that high grafting density is critical for avoiding nonspecific cellular interaction. However, the encapsulation approach resulted in extensive aggregation, as is usually the case, thus limiting the possibility of use in a T1-weighting application. Aggregation was exploited in a recent report where clickable monosaccharides were used to stabilize MNP clusters using complex peptide linkers, which allowed differentiation of cancer cells by quantitative profiling of carbohydrate binding through changes in T2 (local signal suppression). In a prominent recent example, a range of phosphonate-functionalized sugars were used to fully disperse MNPs, and the effect of MNP size on the T1-weighting and hyperthermic properties in vitro were investigated. However, only one sugar per linker was possible, thus limiting the total sugar density. Hence, there is a need for platform technologies that maximize the surface density of functional monosaccharides while allowing full MNP dispersion. Multifunctional polymers have the potential to address the first issue; there have been two recent reports of a “grafting-onto” approach for MNP modification with glycopolymers. However, dispersion proved not to be possible. In the first example, glycosylated polyacrylate was grafted onto silica-coated iron-oxide MNPs. Although cellular uptake of the micron-scale aggregates was confirmed, the magnetic component was quite dilute. In the second example, some improvement in colloidal control was achieved using glucosylated poly(pentafluorostyrene) derivatives to produce suspensions of hydrodynamic size (dhyd) ranging from 100–300 nm. [6] However, the polydispersity index (PDI) from dynamic light scattering was quite high (> 0.22), which suggests non-uniform particles. Herein, we describe the novel combination of a polymer “grafting-from” approach with glycosylation by click chemistry for preparing MNPs with a high surface density of monosaccharides. We hypothesized that this would promote dispersibility and, based on the important role of carbohydrates in cell interaction/recognition, introduce biorecognition. The polymers are synthetic polypeptides and thus rely on natural degradable building blocks (amino acids). This is the first report of glycopeptide-grafted superparamagnetic Fe3O4 nanoparticles (GP-MNP), which exhibit excellent water [*] T. Borase, Dr. T. Ninjbadgar, A. Kapetanakis, Dr. A. Heise, Dr. D. F. Brougham School of Chemical Sciences, Dublin City University Glasnevin, Dublin 9 (Ireland) E-mail: andreas.heise@dcu.ie dermot.brougham@dcu.ie

Drug resistance in cancer - searching for mechanisms, markers and therapeutic agents

Treatment resistance, whether inherent or acquired, is a major problem reducing the activity of conventional and newer, molecularly targeted, cancer drugs. A more complex picture of the causes and contributions of specific forms of resistance is now emerging through application of pharmacological, proteomic and gene expression technologies and we have entered an exciting time where new molecular research tools are being applied not only to characterise the causes of such resistance, but to identify rational new treatments and treatment combinations that are being rapidly translated to clinical evaluations with increasing success. This review outlines many of the contributing causes of resistance to established cytotoxics and to the new breed of molecularly targeted agents, both monoclonal antibodies and small molecules, and the research methods being used to wage war on resistant cancer.

Enhanced in vitro invasiveness and drug resistance with altered gene expression patterns in a human lung carcinoma cell line after pulse selection with anticancer drugs

The human lung carcinoma cell line DLKP was exposed to sequential pulses of 10 commonly used chemotherapeutic drugs (VP‐16, vincristine, taxotere, mitoxantrone, 5‐fluorouracil, methotrexate, CCNU, BCNU, cisplatin and chlorambucil); resulting cell lines exhibited resistance to the selecting agents (ranging approx. 1.5‐ to 36‐fold) and, in some cases, cross‐resistance to methotrexate (approx. 1.4‐ to 22‐fold), vincristine (1.6‐ to 262‐fold), doxorubicin (Adriamycin, approx. 1.1‐ to 33‐fold) and taxotere (approx. 1.1‐ to 36‐fold). Several of the variants displayed collateral sensitivity to cisplatin. A marked increase in in vitro invasiveness and motility was observed with variants pulsed with mitoxantrone, 5‐fluorouracil, methotrexate, BCNU, cisplatin and chlorambucil. There was no significant change in invasiveness of cells pulsed with VP‐16, vincristine, taxotere or CCNU. All of the pulse‐selected variants showed elevated levels of MDR‐1/P‐gp protein by Western blot analysis, although mdr‐1 mRNA levels were not increased (except for DLKP‐taxotere). In DLKP‐taxotere, MRP1 protein levels were also greatly elevated, but mrp1 mRNA levels remained unchanged. BCRP was upregulated in DLKP‐mitoxantrone at both the mRNA and protein levels. Gelatin zymography, Western blot and RT‐PCR showed that DLKP and its variants secreted MMPs 2, 9 and 13. MMP inhibition assays suggested that MMP‐2 plays a more important role than MMPs 9 and 13 in cell invasion of these DLKP drug‐resistant variants in vitro. These results indicate that drug exposure may induce not only resistance but also invasiveness in cancer cells.

Metabolomic studies of human lung carcinoma cell lines using in vitro1H NMR of whole cells and cellular extracts

We report principal component analysis (PCA) of 1H NMR spectra recorded for a group of human lung carcinoma cell lines in culture and 1H NMR analysis of extracts from the same samples. The samples studied were cells of lung tumour origin with different chemotherapy drug resistance patterns. For whole cells, it was found that the statistically significant causes of spectral variation were an increase in the choline and a decrease in the methylene mobile lipid 1H resonance intensities, which correlate with our knowledge of the level of resistance displayed by the different cells. Similarly, in the 1H NMR spectra of the aqueous and lipophilic extracts, significant quantitative differences in the metabolite distributions were apparent, which are consistent with the PCA results. Copyright

Challenges of drug resistance in the management of pancreatic cancer

The current treatment of choice for metastatic pancreatic cancer involves single-agent gemcitabine or a combination of gemcitabine with capecitabine or erlotinib (a tyrosine kinase inhibitor). Only 25–30% of patients respond to this treatment and patients who do respond initially ultimately exhibit disease progression. Median survival for pancreatic cancer patients has reached a plateau due to inherent and acquired resistance to these agents. Key molecular factors implicated in this resistance include: deficiencies in drug uptake, alteration of drug targets, activation of DNA repair pathways, resistance to apoptosis and the contribution of the tumor microenvironment. Moreover, for newer agents including tyrosine kinase inhibitors, overexpression of signaling proteins, mutations in kinase domains, activation of alternative pathways, mutations of genes downstream of the target and/or amplification of the target represent key challenges for treatment efficacy. Here we will review the contribution of known mechanisms and markers of resistance to key pancreatic cancer drug treatments.

A novel panel of protein biomarkers for predicting response to thalidomide-based therapy in newly diagnosed multiple myeloma patients

Multiple myeloma (MM) is a heterogeneous group of disorders both genotypically and phenotypically. Response to thalidomide‐based induction therapy in newly diagnosed patients varies significantly in published clinical trials. Proteomic analysis was performed on 39 newly diagnosed MM patients treated with a thalidomide‐based regimen (22 responders; 17 non‐responders) using immunodepletion, 2‐D DIGE analysis and mass spectrometry. Zinc‐α‐2‐glycoprotein (ZAG), vitamin D‐binding protein (VDB), serum amyloid‐A protein (SAA) and β‐2‐microglobulin (B2M) had statistically significant higher concentrations in non‐responders compared to responders, while haptoglobin (Hp) had a lower concentration. ELISAs were used to validate the candidate protein biomarkers using unfractionated serum from 51 newly diagnosed MM patients (29 responders; 22 non‐responders). Using logistic regression, the best possible area under the curve (AUC) was 0.96 using ZAG, VDB and SAA in combination. Leave‐one‐out‐cross‐validation (LOOCV) indicated an overall predictive accuracy of 84% with associated sensitivity and specificity values of 81.8 and 86.2%, respectively. Subsequently, 16 of 22 thalidomide‐refractory patients successfully achieved complete response or very good partial response using second‐line treatment suggesting that the biomarker profile is specific to thalidomide response rather than identifying patients with MM refractory to all therapies. Using a novel panel of predictive biomarkers, the feasibility of predicting response to thalidomide‐based therapy in previously untreated MM has been demonstrated.

CE-LIF method for the separation of anthracyclines: Application to protein binding analysis in plasma using ultrafiltration

Anthracyclines are chemotherapeutic drugs that are widely used in the treatment of cancers such as lung and ovarian cancers. The simultaneous determination of the anthracyclines, daunorubicin, doxorubicin and epirubicin, was achieved using CE coupled to LIF, with an excitation and emission wavelength of 488 and 560 nm, respectively. Using a borate buffer (105 mM, pH 9.0) and 30% MeOH, a stable and reproducible separation of the three anthracyclines was obtained. The method developed was shown to be capable of monitoring the therapeutic concentrations (50-50 000 ng/mL) of anthracyclines. LODs of 10 ng/mL, calculated at an S/N = 3, were achieved. Using the CE method developed, the in vitro protein binding to plasma was measured by ultrafiltration, and from this investigation the estimated protein binding was determined to be in the range of 77-94%.

Rapid and sensitive liquid chromatography-tandem mass spectrometry for the quantitation of epirubicin and identification of metabolites in biological samples.

A highly sensitive and selective liquid chromatography-mass spectrometry (LC-MS) method has been developed for the determination of epirubicin in serum and cell specimens using daunorubicin as an internal standard. Using atmospheric pressure chemical ionisation (APCI), the epirubicin metabolites were readily distinguishable by their fragmentation pattern in the mass spectrometer. Selected reaction monitoring (SRM) mode was employed for quantitation of epirubicin and the metabolites. Following extraction, chromatography was performed on a C18 column with a mobile phase consisting of water-acetonitrile-formic acid, pH 3.2, with a flow rate of 200mul/min. The limit of detection (LOD) and the limit of quantitation (LOQ) of this method in serum were determined to be 1.0 and 2.5ng/ml, respectively. Linearity of the method was verified over the concentration range of 2.5-2000ng/ml, with a high correlation coefficient (R(2)>/=0.998). For the extraction procedure, an aliquot of 500mul serum, spiked with internal standard, was extracted using a chloroform-2-isopropanol (2:1, v/v) mixture. The method has been applied to the analysis of epirubicin in cancer cell samples and the identification of known and unknown metabolites in clinical trial patient serum samples.

Synthesis of indomethacin analogues for evaluation as modulators of MRP activity

Synthesis of a range of indomethacin analogues, required for investigation in combination toxicity assays, bearing both N-benzyl and N-benzoyl groups, is described.