Bruce C. Baguley

From amsacrine to DACA (N-[2-(dimethylamino)ethyl]acridine-4-carboxamide): Selectivity for topoisomerases I and II among acridine derivatives

A number of acridine derivatives, including the clinical antileukaemia agent amsacrine and the experimental agent DACA (N-[2-(dimethylamino)ethyl]acridine-4-carboxamide), target the enzyme topoisomerase II. We demonstrate here that DACA induces DNA cleavage in the presence of topoisomerase I as well as of topoisomerase II. We also investigate a series of acridine derivatives which link amsacrine to DACA in terms of DNA binding, topoisomerase poisoning and biological activity. The presence of an acridine 4-linked N-2-(dimethylamino)ethyl group provides both a pronounced G-C preference for DNA binding and activity towards topoisomerase I. The removal of the anilino side chain of amsacrine, in combination with the presence of the N-2-(dimethylamino)ethyl group, provides in vitro biological activity against atypical multidrug resistant leukaemia lines with low topoisomerase II activity. Among these compounds, suppression of the ionisation of the acridine nitrogen to produce the compound DACA is associated with experimental activity against solid tumours. The addition of an acridine 2-chloro substituent to DACA suppresses the stimulation of topoisomerase II-dependent DNA cleavage but increases stimulation of topoisomerase I cleavage. 2-Substitution also increases activity against the atypical multidrug resistant cell lines. Overall, the results suggest that augmentation of topoisomerase I-dependent activity in this series by appropriate chemical substitution in this series leads to circumvention of topoisomerase II-mediated multidrug resistance.

Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists, 1994: PHARMACOKINETIC/CYTOKINETIC PRINCIPLES IN THE CHEMOTHERAPY OF SOLID TUMOURS

1. The solid tumour has various properties which tend to minimize the effects of a cytotoxic agent; the low vascular density of tumours, in particular, limits the diffusion of many anti‐tumour drugs.

Multiple Isoforms of ANRIL in Melanoma Cells: Structural Complexity Suggests Variations in Processing

The long non-coding RNA ANRIL, antisense to the CDKN2B locus, is transcribed from a gene that encompasses multiple disease-associated polymorphisms. Despite the identification of multiple isoforms of ANRIL, expression of certain transcripts has been found to be tissue-specific and the characterisation of ANRIL transcripts remains incomplete. Several functions have been associated with ANRIL. In our judgement, studies on ANRIL functionality are premature pending a more complete appreciation of the profusion of isoforms. We found differential expression of ANRIL exons, which indicates that multiple isoforms exist in melanoma cells. In addition to linear isoforms, we identified circular forms of ANRIL (circANRIL). Further characterisation of circANRIL in two patient-derived metastatic melanoma cell lines (NZM7 and NZM37) revealed the existence of a rich assortment of circular isoforms. Moreover, in the two melanoma cell lines investigated, the complements of circANRIL isoforms were almost completely different. Novel exons were also discovered. We also found the family of linear ANRIL was enriched in the nucleus, whilst the circular isoforms were enriched in the cytoplasm and they differed markedly in stability. With respect to the variable processing of circANRIL species, bioinformatic analysis indicated that intronic Arthrobacter luteus (Alu) restriction endonuclease inverted repeats and exon skipping were not involved in selection of back-spliced exon junctions. Based on our findings, we hypothesise that “ANRIL” has wholly distinct dual sets of functions in melanoma. This reveals the dynamic nature of the locus and constitutes a basis for investigating the functions of ANRIL in melanoma.

Synthesis and biological evaluation of sulfonamide analogues of the phosphatidylinositol 3-kinase inhibitor ZSTK474

Replacement of one of the morpholine groups of the phosphatidylinositol 3-kinase (PI3K) inhibitor ZSTK474 (1) with sulfonamide containing substituents produced a new class of active and potent PI3Kα inhibitors. Solubility issues prevented all but the 6-amino derivative 17 from being evaluated in vivo, but the clear activity of this compound demonstrated that this class of PI3K inhibitor shows great promise.

Endocrine Therapy of Estrogen Receptor-Positive Breast Cancer Cells: Early Differential Effects on Stem Cell Markers

Introduction Endocrine therapy of breast cancer, which either deprives cancer tissue of estrogen or prevents estrogen pathway signaling, is the most common treatment after surgery and radiotherapy. We have previously shown for the estrogen-responsive MCF-7 cell line that exposure to tamoxifen, or deprivation of estrogen, leads initially to inhibition of cell proliferation, followed after several months by the emergence of resistant sub-lines that are phenotypically different from the parental line. We examined the early responses of MCF-7 cells following either exposure to 4-hydroxytamoxifen or deprivation of estrogen for periods of 2u2009days–4u2009weeks. Methods Endocrine-sensitive or -resistant breast cancer cell lines were used to examine the expression of the stem cell gene SOX2, and the Wnt effector genes AXIN2 and DKK1 using quantitative PCR analysis. Breast cancer cell lines were used to assess the anti-proliferative effects (as determined by IC50 values) of Wnt pathway inhibitors LGK974 and IWP-2. Results Hormone therapy led to time-dependent increases of up to 10-fold in SOX2 expression, up to threefold in expression of the Wnt target genes AXIN2 and DKK1, and variable changes in NANOG and OCT4 expression. The cells also showed increased mammosphere formation and increased CD24 surface protein expression. Some but not all hormone-resistant MCF-7 sub-lines, emerging after long-term hormonal stress, showed up to 50-fold increases in SOX2 expression and smaller increases in AXIN2 and DKK1 expression. However, the increase in Wnt target gene expression was not accompanied by an increase in sensitivity to Wnt pathway inhibitors LGK974 and IWP-2. A general trend of lower IC50 values was observed in 3-dimensional spheroid culture conditions (which allowed enrichment of cells with cancer stem cell phenotype) relative to monolayer cultures. The endocrine-resistant cell lines showed no significant increase in sensitivity to Wnt inhibitors. Conclusion Hormone treatment of cultured MCF-7 cells leads within 2u2009days to increased expression of components of the SOX2 and Wnt pathways and to increased potential for mammosphere formation. We suggest that these responses are indicative of early adaptation to endocrine stress with features of stem cell character and that this facilitates the survival of emerging hormone-resistant cell populations.

The Genes of Life and Death: A Potential Role for Placental-Specific Genes in Cancer

The placenta invades the adjacent uterus and controls the maternal immune system, like a cancer invades surrounding organs and suppresses the local immune response. Intriguingly, placental and cancer cells are globally hypomethylated and share an epigenetic phenomenon that is not well understood – they fail to silence repetitive DNA sequences (retrotransposons) that are silenced (methylated) in healthy somatic cells. In the placenta, hypomethylation of retrotransposons has facilitated the evolution of new genes essential for placental function. In cancer, hypomethylation is thought to contribute to activation of oncogenes, genomic instability, and retrotransposon unsilencing; the latter, we postulate, is possibly the most important consequence. Activation of placental retrotransposon‐derived genes in cancer underpins our hypothesis that hypomethylation of these genes drives cancer cell invasion. This alludes to an interesting paradox, that while placental retrotransposon‐derived genes are essential for promoting early hominid life, the same genes promote disease‐susceptibility and death through cancer.

Optimization of Weight Ratio for DSPE-PEG/TPGS Hybrid Micelles to Improve Drug Retention and Tumor Penetration

PurposeTo enhance therapeutic efficacy and prevent phlebitis caused by Asulacrine (ASL) precipitation post intravenous injection, ASL-loaded hybrid micelles with size below 40xa0nm were developed to improve drug retention and tumor penetration.MethodsASL-micelles were prepared using different weight ratios of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethyleneglycol-2000 (DSPE-PEG2000) and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) polymers. Stability of micelles was optimized in terms of critical micelle concentration (CMC) and drug release properties. The encapsulation efficiency (EE) and drug loading were determined using an established dialysis-mathematic fitting method. Multicellular spheroids (MCTS) penetration and cytotoxicity were investigated on MCF-7 cell line. Pharmacokinetics of ASL-micelles was evaluated in rats with ASL-solution as control.ResultsThe ASL-micelles prepared with DSPE-PEG2000 and TPGS (1:1, w/w) exhibited small size (~18.5xa0nm), higher EE (~94.12%), better sustained in vitro drug release with lower CMC which may be ascribed to the interaction between drug and carriers. Compared to free ASL, ASL-micelles showed better MCTS penetration capacity and more potent cytotoxicity. Pharmacokinetic studies demonstrated that the half-life and AUC values of ASL-micelles were approximately 1.37-fold and 3.49-fold greater than that of free ASL.ConclusionsThe optimized DSPE-PEG2000/TPGS micelles could serve as a promising vehicle to improve drug retention and penetration in tumor.

Correction: Sarkar, D., et al. Multiple Isoforms of ANRIL in Melanoma Cells: Structural Complexity Suggests Variations in Processing. Int. J. Mol. Sci. 2017, 18, 1378

Debina Sarkar 1,2, Ali Oghabian 3, Pasani K. Bodiyabadu 1,2, Wayne R. Joseph 1, Euphemia Y. Leung 1,2, Graeme J. Finlay 1,2,*, Bruce C. Baguley 1 and Marjan E. Askarian-Amiri 1,2,* ID 1 Auckland Cancer Society Research Centre, University of Auckland, Faculty of Medical and Health Sciences, University of Auckland, 85 Park Rd. Grafton, 1023 Auckland, New Zealand; d.sarkar@auckland.ac.nz (D.S.); pbod004@aucklanduni.ac.nz (P.K.B.); w.joseph@auckland.ac.nz (W.R.J.); e.leung@auckland.ac.nz (E.Y.L.); b.baguley@auckland.ac.nz (B.C.B.) 2 Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, University of Auckland, 85 Park Rd. Grafton, 1023 Auckland, New Zealand 3 Institute of Biotechnology, P.O. Box 56 (Viikinkaari 5), University of Helsinki, FI-00014 Helsinki, Finland; ali.oghabian@helsinki.fi * Correspondence: g.finlay@auckland.ac.nz (G.J.F.); m.askarian-amiri@auckland.ac.nz (M.E.A.-A.); Tel.: +64-9-923-6092 (G.J.F.); +64-9-923-6803 (M.E.A.-A.); Fax: +64-9-373-7502 (G.J.F. & M.E.A.-A.)

Multiseed liposomal drug delivery system using micelle gradient as driving force to improve amphiphilic drug retention and its anti-tumor efficacy

Abstract To improve drug retention in carriers for amphiphilic asulacrine (ASL), a novel active loading method using micelle gradient was developed to fabricate the ASL-loaded multiseed liposomes (ASL-ML). The empty ML were prepared by hydrating a thin film with empty micelles. Then the micelles in liposomal compartment acting as ‘micelle pool’ drove the drug to be loaded after the outer micelles were removed. Some reasoning studies including critical micelle concentration (CMC) determination, influencing factors tests on entrapment efficiency (EE), structure visualization, and drug release were carried out to explore the mechanism of active loading, ASL location, and the structure of ASL-ML. Comparisons were made between pre-loading and active loading method. Finally, the extended drug retention capacity of ML was evaluated through pharmacokinetic, drug tissue irritancy, and in vivo anti-tumor activity studies. Comprehensive results from fluorescent and transmission electron microscope (TEM) observation, encapsulation efficiency (EE) comparison, and release studies demonstrated the formation of ML-shell structure for ASL-ML without inter-carrier fusion. The location of drug mainly in inner micelles as well as the superiority of post-loading to the pre-loading method , in which drug in micelles shifted onto the bilayer membrane was an additional positive of this delivery system. It was observed that the drug amphiphilicity and interaction of micelles with drug were the two prerequisites for this active loading method. The extended retention capacity of ML has been verified through the prolonged half-life, reduced paw-lick responses in rats, and enhanced tumor inhibition in model mice. In conclusion, ASL-ML prepared by active loading method can effectively load drug into micelles with expected structure and improve drug retention.