Publications by authors named "Bruce C Baguley"

137 Publications

Comparison of Global DNA Methylation Patterns in Human Melanoma Tissues and Their Derivative Cell Lines.

Cancers (Basel) 2021 Apr 28;13(9). Epub 2021 Apr 28.

Department of Pathology, Otago Medical School-Dunedin Campus, University of Otago, Dunedin 9054, New Zealand.

DNA methylation is a heritable epigenetic mark that is fundamental to mammalian development. Aberrant DNA methylation is an epigenetic hallmark of cancer cells. Cell lines are a critical in vitro model and very widely used to unravel mechanisms of cancer cell biology. However, limited data are available to assess whether DNA methylation patterns in tissues are retained when cell lines are established. Here, we provide the first genome-scale sequencing-based methylation map of metastatic melanoma tumour tissues and their derivative cell lines. We show that DNA methylation profiles are globally conserved in vitro compared to the tumour tissue of origin. However, we identify sites that are consistently hypermethylated in cell lines compared to their tumour tissue of origin. The genes associated with these common differentially methylated regions are involved in cell metabolism, cell cycle and apoptosis and are also strongly enriched for the H3K27me3 histone mark and PRC2 complex-related genes. Our data indicate that although global methylation patterns are similar between tissues and cell lines, there are site-specific epigenomic differences that could potentially impact gene expression. Our work provides a valuable resource for identifying false positives due to cell culture and for better interpretation of cancer epigenetics studies in the future.
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http://dx.doi.org/10.3390/cancers13092123DOI Listing
April 2021

Genome-wide DNA methylation and RNA expression differences correlate with invasiveness in melanoma cell lines.

Epigenomics 2021 Apr 30;13(8):577-598. Epub 2021 Mar 30.

Department of Pathology, Otago Medical School - Dunedin Campus, University of Otago, Dunedin 9054, New Zealand.

The aim of this study was to investigate the role of DNA methylation in invasiveness in melanoma cells. : The authors carried out genome-wide transcriptome (RNA sequencing) and reduced representation bisulfite sequencing methylome profiling between noninvasive (n = 4) and invasive melanoma cell lines (n = 5).  The integration of differentially expressed genes and differentially methylated fragments (DMFs) identified 12 DMFs (two in , one in , two in , one in , one in , one in and four in ) that overlapped with either differentially expressed genes (eight DMFs and six genes) or cis-targets of lncRNAs (five DMFs associated with cis-targets and four differentially expressed lncRNAs).  DNA methylation changes are associated with a number of transcriptional differences observed in noninvasive and invasive phenotypes in melanoma.
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http://dx.doi.org/10.2217/epi-2020-0440DOI Listing
April 2021

Diverse mechanisms activate the PI 3-kinase/mTOR pathway in melanomas: implications for the use of PI 3-kinase inhibitors to overcome resistance to inhibitors of BRAF and MEK.

BMC Cancer 2021 Feb 6;21(1):136. Epub 2021 Feb 6.

Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand.

Background: The PI 3-kinase (PI3K) pathway has been implicated as a target for melanoma therapy.

Methods: Given the high degree of genetic heterogeneity in melanoma, we sought to understand the breadth of variation in PI3K signalling in the large NZM panel of early passage cell lines developed from metastatic melanomas.

Results: We find the vast majority of lines show upregulation of this pathway, and this upregulation is achieved by a wide range of mechanisms. Expression of all class-IA PI3K isoforms was readily detected in these cell lines. A range of genetic changes in different components of the PI3K pathway was seen in different lines. Coding variants or amplification were identified in the PIK3CA gene, and amplification of the PK3CG gene was common. Deletions in the PIK3R1 and PIK3R2 regulatory subunits were also relatively common. Notably, no genetic variants were seen in the PIK3CD gene despite p110δ being expressed in many of the lines. Genetic variants were detected in a number of genes that encode phosphatases regulating the PI3K signalling, with reductions in copy number common in PTEN, INPP4B, INPP5J, PHLLP1 and PHLLP2 genes. While the pan-PI3K inhibitor ZSTK474 attenuated cell growth in all the lines tested, isoform-selective inhibition of p110α and p110δ inhibited cell growth in only a subset of the lines and the inhibition was only partial. This suggests that functional redundancy exists between PI3K isoforms. Furthermore, while ZSTK474 was initially effective in melanoma cells with induced resistance to vemurafenib, a subset of these cell lines concurrently developed partial resistance to PI3K inhibition. Importantly, mTOR-selective or mTOR/PI3K dual inhibitors effectively inhibited cell growth in all the lines, including those already resistant to BRAF inhibitors and ZSTK474.

Conclusions: Overall, this indicates a high degree of diversity in the way the PI3K pathway is activated in different melanoma cell lines and that mTOR is the most effective point for targeting the growth via the PI3K pathway across all of these cell lines.
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http://dx.doi.org/10.1186/s12885-021-07826-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866738PMC
February 2021

DNA-Binding Anticancer Drugs: One Target, Two Actions.

Molecules 2021 Jan 21;26(3). Epub 2021 Jan 21.

Auckland Cancer Society Research Centre, Faculty of Medical Sciences, The University of Auckland, Auckland 1023, New Zealand.

Amsacrine, an anticancer drug first synthesised in 1970 by Professor Cain and colleagues, showed excellent preclinical activity and underwent clinical trial in 1978 under the auspices of the US National Cancer Institute, showing activity against acute lymphoblastic leukaemia. In 1984, the enzyme DNA topoisomerase II was identified as a molecular target for amsacrine, acting to poison this enzyme and to induce DNA double-strand breaks. One of the main challenges in the 1980s was to determine whether amsacrine analogues could be developed with activity against solid tumours. A multidisciplinary team was assembled in Auckland, and Professor Denny played a leading role in this approach. Among a large number of drugs developed in the programme, -[2-(dimethylamino)-ethyl]-acridine-4-carboxamide (DACA), first synthesised by Professor Denny, showed excellent activity against a mouse lung adenocarcinoma. It underwent clinical trial, but dose escalation was prevented by ion channel toxicity. Subsequent work led to the DACA derivative SN 28049, which had increased potency and reduced ion channel toxicity. Mode of action studies suggested that both amsacrine and DACA target the enzyme DNA topoisomerase II but with a different balance of cellular consequences. As primarily a topoisomerase II poison, amsacrine acts to turn the enzyme into a DNA-damaging agent. As primarily topoisomerase II catalytic inhibitors, DACA and SN 28049 act to inhibit the segregation of daughter chromatids during anaphase. The balance between these two actions, one cell cycle phase specific and the other nonspecific, together with pharmacokinetic, cytokinetic and immunogenic considerations, provides links between the actions of acridine derivatives and anthracyclines such as doxorubicin. They also provide insights into the action of cytotoxic DNA-binding drugs.
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http://dx.doi.org/10.3390/molecules26030552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866126PMC
January 2021

Genomic and signalling pathway characterization of the NZM panel of melanoma cell lines: A valuable model for studying the impact of genetic diversity in melanoma.

Pigment Cell Melanoma Res 2021 01 4;34(1):136-143. Epub 2020 Jul 4.

Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand.

Melanoma is a disease associated with a very high mutation burden and thus the possibility of a diverse range of oncogenic mechanisms that allow it to evade therapeutic interventions and the immune system. Here, we describe the characterization of a panel of 102 cell lines from metastatic melanomas (the NZM lines), including using whole-exome and RNA sequencing to analyse genetic variants and gene expression changes in a subset of this panel. Lines possessing all major melanoma genotypes were identified, and hierarchical clustering of gene expression profiles revealed four broad subgroups of cell lines. Immunogenotyping identified a range of HLA haplotypes as well as expression of neoantigens and cancer-testis antigens in the lines. Together, these characteristics make the NZM panel a valuable resource for cell-based, immunological and xenograft studies to better understand the diversity of melanoma biology and the responses of melanoma to therapeutic interventions.
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http://dx.doi.org/10.1111/pcmr.12908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7818249PMC
January 2021

Pyruvate anaplerosis is a mechanism of resistance to pharmacological glutaminase inhibition in triple-receptor negative breast cancer.

BMC Cancer 2020 May 25;20(1):470. Epub 2020 May 25.

Auckland Cancer Society Research Centre, School of Medical Sciences, Faculty of Medical and Health Sciences University of Auckland, Private Bag, Auckland, 92019, New Zealand.

Background: Glutamine serves as an important nutrient with many cancer types displaying glutamine dependence. Following cellular uptake glutamine is converted to glutamate in a reaction catalysed by mitochondrial glutaminase. This glutamate has many uses, including acting as an anaplerotic substrate (via alpha-ketoglutarate) to replenish TCA cycle intermediates. CB-839 is a potent, selective, orally bioavailable inhibitor of glutaminase that has activity in Triple receptor-Negative Breast Cancer (TNBC) cell lines and evidence of efficacy in advanced TNBC patients.

Methods: A panel of eleven breast cancer cell lines was used to investigate the anti-proliferative effects of the glutaminase inhibitors CB-839 and BPTES in different types of culture medium, with or without additional pyruvate supplementation. The abundance of the TCA cycle intermediate fumarate was quantified as a measure if TCA cycle anaplerosis. Pyruvate secretion by TNBC cultures was then assessed with or without AZD3965, a monocarboxylate transporter 1 (MCT1) inhibitor. Finally, two dimensional (2D) monolayer and three dimensional (3D) spheroid assays were used to compare the effect of microenvironmental growth conditions on CB-839 activity.

Results: The anti-proliferative activity of CB-839 in a panel of breast cancer cell lines was similar to published reports, but with a major caveat; growth inhibition by CB-839 was strongly attenuated in culture medium containing pyruvate. This pyruvate-dependent attenuation was also observed with a related glutaminase inhibitor, BPTES. Studies demonstrated that exogenous pyruvate acted as an anaplerotic substrate preventing the decrease of fumarate in CB-839-treated conditions. Furthermore, endogenously produced pyruvate secreted by TNBC cell lines was able to act in a paracrine manner to significantly decrease the sensitivity of recipient cells to glutaminase inhibition. Suppression of pyruvate secretion using the MCT1 inhibitor AZD3965, antagonised this paracrine effect and increased CB-839 activity. Finally, CB-839 activity was significantly compromised in 3D compared with 2D TNBC culture models, suggesting that 3D microenvironmental features impair glutaminase inhibitor responsiveness.

Conclusion: This study highlights the potential influence that both circulating and tumour-derived pyruvate can have on glutaminase inhibitor efficacy. Furthermore, it highlights the benefits of 3D spheroid cultures to model the features of the tumour microenvironment and improve the in vitro investigation of cancer metabolism-targeted therapeutics.
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http://dx.doi.org/10.1186/s12885-020-06885-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7333265PMC
May 2020

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

Bioorg Med Chem 2019 04 25;27(8):1529-1545. Epub 2019 Feb 25.

Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand. Electronic address:

Replacing one of the morpholine groups of the phosphatidylinositol 3-kinase (PI3K) inhibitor ZSTK474 with a variety of sulfonamide-linked solubilizing substituents produced a new class of active and potent PI3Kα inhibitors, with several derivatives demonstrating high PI3Kα enzyme potency and good cellular potency in two human derived cell lines. The overall results suggest a preference for linear and somewhat flexible solubilizing functions. From this series, compound 16, also known as SN32976, was selected for advanced preclinical evaluation.
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http://dx.doi.org/10.1016/j.bmc.2019.02.050DOI Listing
April 2019

Derivation of Breast Cancer Cell Lines Under Physiological (5%) Oxygen Concentrations.

Front Oncol 2018 12;8:425. Epub 2018 Oct 12.

Auckland Cancer Society Research Centre, University of Auckland, Auckland, New Zealand.

Most human breast cancer cell lines currently in use were developed and are cultured under ambient (21%) oxygen conditions. While this is convenient in practical terms, higher ambient oxygen could increase oxygen radical production, potentially modulating signaling pathways. We have derived and grown a series of four human breast cancer cell lines under 5% oxygen, and have compared their properties to those of established breast cancer lines growing under ambient oxygen. Cell lines were characterized in terms of appearance, cellular DNA content, mutation spectrum, hormone receptor status, pathway utilization and drug sensitivity. Three of the four lines (NZBR1, NZBR2, and NZBR4) were triple negative (ER-, PR-, HER2-), with NZBR1 also over-expressing EGFR. NZBR3 was HER2+ and ER+ and also over-expressed EGFR. Cell lines grown in 5% oxygen showed increased expression of the hypoxia-inducible factor 1 (HIF-1) target gene carbonic anhydrase 9 () and decreased levels of ROS. As determined by protein phosphorylation, NZBR1 showed low AKT pathway utilization while NZBR2 and NZBR4 showed low p70S6K and rpS6 pathway utilization. The lines were characterized for sensitivity to 7-hydroxytamoxifen, doxorubicin, paclitaxel, the PI3K inhibitor BEZ235 and the HER inhibitors lapatinib, afatinib, dacomitinib, and ARRY-380. In some cases they were compared to established breast cancer lines. Of particular note was the high sensitivity of NZBR3 to HER inhibitors. The spectrum of mutations in the NZBR lines was generally similar to that found in commonly used breast cancer cell lines but mutations were absent and mutations in , and , which have not been reported in other breast cancer lines, were detected. The results suggest that the properties of cell lines developed under low oxygen conditions (5% O) are similar to those of commonly used breast cancer cell lines. Although reduced ROS production and increased HIF-1 activity under 5% oxygen can potentially influence experimental outcomes, no difference in sensitivity to estrogen or doxorubicin was observed between cell lines cultured in 5 vs. 21% oxygen.
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http://dx.doi.org/10.3389/fonc.2018.00425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6194255PMC
October 2018

Correction: Sarkar, D., et al. Multiple Isoforms of in Melanoma Cells: Structural Complexity Suggests Variations in Processing. 2017, , 1378.

Int J Mol Sci 2018 05 2;19(5). Epub 2018 May 2.

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.

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http://dx.doi.org/10.3390/ijms19051343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983800PMC
May 2018

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

Drug Deliv 2018 Nov;25(1):611-622

a Department of Pharmaceutics , China Pharmaceutical University , Nanjing , PR China.

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.
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http://dx.doi.org/10.1080/10717544.2018.1440669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058678PMC
November 2018

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

Pharm Res 2018 01 4;35(1):13. Epub 2018 Jan 4.

Department of Pharmaceutics, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009, People's Republic of China.

Purpose: To enhance therapeutic efficacy and prevent phlebitis caused by Asulacrine (ASL) precipitation post intravenous injection, ASL-loaded hybrid micelles with size below 40 nm were developed to improve drug retention and tumor penetration.

Methods: ASL-micelles were prepared using different weight ratios of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethyleneglycol-2000 (DSPE-PEG) 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.

Results: The ASL-micelles prepared with DSPE-PEG and TPGS (1:1, w/w) exhibited small size (~18.5 nm), 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.

Conclusions: The optimized DSPE-PEG/TPGS micelles could serve as a promising vehicle to improve drug retention and penetration in tumor.
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http://dx.doi.org/10.1007/s11095-017-2340-yDOI Listing
January 2018

The Genes of Life and Death: A Potential Role for Placental-Specific Genes in Cancer: Active retrotransposons in the placenta encode unique functional genes that may also be used by cancer cells to promote malignancy.

Bioessays 2017 11 4;39(11). Epub 2017 Oct 4.

Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin 9054, New Zealand.

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.
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http://dx.doi.org/10.1002/bies.201700091DOI Listing
November 2017

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

Bioorg Med Chem 2017 10 20;25(20):5859-5874. Epub 2017 Sep 20.

Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bmc.2017.09.025DOI Listing
October 2017

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

Front Oncol 2017 4;7:184. Epub 2017 Sep 4.

Auckland Cancer Society Research Centre, University of Auckland, Auckland, New Zealand.

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 2 days-4 weeks.

Methods: Endocrine-sensitive or -resistant breast cancer cell lines were used to examine the expression of the stem cell gene , and the Wnt effector genes and using quantitative PCR analysis. Breast cancer cell lines were used to assess the anti-proliferative effects (as determined by IC values) of Wnt pathway inhibitors LGK974 and IWP-2.

Results: Hormone therapy led to time-dependent increases of up to 10-fold in expression, up to threefold in expression of the Wnt target genes and , and variable changes in and 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 expression and smaller increases in and 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 IC 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 2 days to increased expression of components of the 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.
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http://dx.doi.org/10.3389/fonc.2017.00184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5591432PMC
September 2017

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

Int J Mol Sci 2017 Jun 27;18(7). Epub 2017 Jun 27.

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.

The long non-coding RNA , antisense to the locus, is transcribed from a gene that encompasses multiple disease-associated polymorphisms. Despite the identification of multiple isoforms of , expression of certain transcripts has been found to be tissue-specific and the characterisation of transcripts remains incomplete. Several functions have been associated with In our judgement, studies on functionality are premature pending a more complete appreciation of the profusion of isoforms. We found differential expression of exons, which indicates that multiple isoforms exist in melanoma cells. In addition to linear isoforms, we identified circular forms of (). Further characterisation of 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 isoforms were almost completely different. Novel exons were also discovered. We also found the family of linear 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 species, bioinformatic analysis indicated that intronic (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 "" 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 in melanoma.
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http://dx.doi.org/10.3390/ijms18071378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5535871PMC
June 2017

PIK3CA-mutated melanoma cells rely on cooperative signaling through mTORC1/2 for sustained proliferation.

Pigment Cell Melanoma Res 2017 05 20;30(3):353-367. Epub 2017 Apr 20.

Helen Diller Family Comprehensive Cancer Center, Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA.

Malignant conversion of BRAF- or NRAS-mutated melanocytes into melanoma cells can be promoted by PI3'-lipid signaling. However, the mechanism by which PI3'-lipid signaling cooperates with mutationally activated BRAF or NRAS has not been adequately explored. Using human NRAS- or BRAF-mutated melanoma cells that co-express mutationally activated PIK3CA, we explored the contribution of PI3'-lipid signaling to cell proliferation. Despite mutational activation of PIK3CA, melanoma cells were more sensitive to the biochemical and antiproliferative effects of broader spectrum PI3K inhibitors than to an α-selective PI3K inhibitor. Combined pharmacological inhibition of MEK1/2 and PI3K signaling elicited more potent antiproliferative effects and greater inhibition of the cell division cycle compared to single-agent inhibition of either pathway alone. Analysis of signaling downstream of MEK1/2 or PI3K revealed that these pathways cooperate to regulate cell proliferation through mTORC1-mediated effects on ribosomal protein S6 and 4E-BP1 phosphorylation in an AKT-dependent manner. Although PI3K inhibition resulted in cytostatic effects on xenografted NRAS /PIK3CA melanoma, combined inhibition of MEK1/2 plus PI3K elicited significant melanoma regression. This study provides insights as to how mutationally activated PIK3CA acts in concert with MEK1/2 signaling to cooperatively regulate mTORC1/2 to sustain PIK3CA-mutated melanoma proliferation.
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http://dx.doi.org/10.1111/pcmr.12586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5411292PMC
May 2017

Novel pyrazolo[1,5-a]pyridines with improved aqueous solubility as p110α-selective PI3 kinase inhibitors.

Bioorg Med Chem Lett 2017 01 25;27(2):187-190. Epub 2016 Nov 25.

Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand; Department of Molecular Medicine and Pathology, School of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.

As part of our investigation into pyrazolo[1,5-a]pyridines as novel p110α selective PI3 kinase inhibitors, we report a range of analogues with improved aqueous solubility by the addition of a basic amine. The compounds demonstrated comparable p110α potency and selectivity to earlier compounds but with up to 1000× greater aqueous solubility, as the hydrochloride salts. The compounds also displayed good activity in a cellular assay of PI3 kinase activity.
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http://dx.doi.org/10.1016/j.bmcl.2016.11.078DOI Listing
January 2017

ZFAS1: a long noncoding RNA associated with ribosomes in breast cancer cells.

Biol Direct 2016 11 21;11(1):62. Epub 2016 Nov 21.

Auckland Cancer Society Research Centre, University of Auckland, 85 Park Rd, Grafton, Auckland, 1023, New Zealand.

Background: Most of the eukaryotic genome is transcribed, yielding a complex network of transcripts including thousands of lncRNAs that generally lack protein coding potential. However, only a small percentage of these molecules has been functionally characterised, and discoveries of specific functions demonstrate layers of complexity. A large percentage of lncRNAs is located in the cytoplasm, associated with ribosomes but the function of the majority of these transcripts is unclear. The current study analyses putative mechanisms of action of the lncRNA species member ZFAS1 that was initially discovered by microarray analysis of murine tissues undergoing mammary gland development. As developmental genes are often deregulated in cancer, here we have studied its function in breast cancer cell lines.

Results: Using human breast cancer cell lines, ZFAS1 was found to be expressed in all cell lines tested, albeit at different levels of abundance. Following subcellular fractionation, human ZFAS1 was found in both nucleus and cytoplasm (as is the mouse orthologue) in an isoform-independent manner. Sucrose gradients based on velocity sedimentation were utilised to separate the different components of total cell lysate, and surprisingly ZFAS1 was primarily co-localised with light polysomes. Further investigation into ribosome association through subunit dissociation studies showed that ZFAS1 was predominantly associated with the 40S small ribosomal subunit. The expression levels of ZFAS1 and of mRNAs encoding several ribosomal proteins that have roles in ribosome assembly, production and maturation were tightly correlated. ZFAS1 knockdown significantly reduced RPS6 phosphorylation.

Conclusion: A large number of lncRNAs associate with ribosomes but the function of the majority of these lncRNAs has not been elucidated. The association of the lncRNA ZFAS1 with a subpopulation of ribosomes and the correlation with expression of mRNAs for ribosomal proteins suggest a ribosome-interacting mechanism pertaining to their assembly or biosynthetic activity. ZFAS1 may represent a new class of lncRNAs which associates with ribosomes to regulate their function.

Reviewers: This article was reviewed by Christine Vande Velde, Nicola Aceto and Haruhiko Siomi.
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http://dx.doi.org/10.1186/s13062-016-0165-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5117590PMC
November 2016

Optimization of the formation of embedded multicellular spheroids of MCF-7 cells: How to reliably produce a biomimetic 3D model.

Anal Biochem 2016 Dec 5;515:47-54. Epub 2016 Oct 5.

Department of Pharmaceutics, China Pharmaceutical University, Nanjing, 210009, PR China. Electronic address:

To obtain a multicellular MCF-7 spheroid model to mimic the three-dimensional (3D) of tumors, the microwell liquid overlay (A) and hanging-drop/agar (B) methods were first compared for their technical parameters. Then a method for embedding spheroids within collagen was optimized. For method A, centrifugation assisted cells form irregular aggregates but not spheroids. For method B, an extended sedimentation period of over 24 h for cell suspensions and increased viscosity of the culture medium using methylcellulose were necessary to harvest a dense and regular cell spheroid. When the number was less than 5000 cells/drop, embedded spheroids showed no tight cores and higher viability than the unembedded. However, above 5000 cells/drop, cellular viability of embedded spheroids was not significantly different from unembedded spheroids and cells invading through the collagen were in a sun-burst pattern with tight cores. Propidium Iodide staining indicated that spheroids had necrotic cores. The doxorubicin cytotoxicity demonstrated that spheroids were less susceptible to DOX than their monolayer cells. A reliable and reproducible method for embedding spheroids using the hanging-drop/agarose method within collagen is described herein. The cell culture model can be used to guide experimental manipulation of 3D cell cultures and to evaluate anticancer drug efficacy.
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http://dx.doi.org/10.1016/j.ab.2016.10.004DOI Listing
December 2016

Selected GRIN2A mutations in melanoma cause oncogenic effects that can be modulated by extracellular glutamate.

Cell Calcium 2016 12 14;60(6):384-395. Epub 2016 Sep 14.

Department of Molecular Medicine and Pathology, University of Auckland, Private Bag 92019, Auckland, New Zealand; LabPlus Haematology, Auckland City Hospital, Private Bag 92024, Auckland, New Zealand. Electronic address:

GRIN2A mutations are frequent in melanoma tumours but their role in disease is not well understood. GRIN2A encodes a modulatory subunit of the N-methyl-d-aspartate receptor (NMDAR). We hypothesized that certain GRIN2A mutations increase NMDAR function and support melanoma growth through oncogenic effects. This hypothesis was tested using 19 low-passage melanoma cell lines, four of which carried novel missense mutations in GRIN2A that we previously reported. We examined NMDAR expression, function of a calcium ion (Ca) channel and its contribution to cell growth using pharmacological modulators; findings were correlated with the presence or absence of GRIN2A mutations. We found that NMDAR expression was low in all melanoma cell lines, independent of GRIN2A mutations. In keeping with this, NMDAR-mediated Ca influx and its contribution to cell proliferation were weak in most cell lines. However, certain GRIN2A mutations and culture media with lower glutamate levels enhanced NMDAR effects on cell growth and invasion. The main finding was that G762E was associated with higher glutamate-mediated Ca influx and stronger NMDAR contribution to cell proliferation, compared with wild-type GRIN2A and other GRIN2A mutations. The pro-invasive phenotype of mutated cell lines was increased in culture medium containing less glutamate, implying environmental modulation of mutation effects. In conclusion, NMDAR ion channel function is low in cultured melanoma cells but supports cell proliferation and invasion. Selected GRIN2A mutations, such as G762E, are associated with oncogenic consequences that can be modulated by extracellular glutamate. Primary cultures may be better suited to determine the role of the NMDAR in melanoma in vivo.
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http://dx.doi.org/10.1016/j.ceca.2016.09.003DOI Listing
December 2016

Signaling Pathways in Melanogenesis.

Int J Mol Sci 2016 Jul 15;17(7). Epub 2016 Jul 15.

Department of Molecular Medicine and Pathology, University of Auckland, Faculty of Medical and Health Sciences, University of Auckland, 85 Park Rd. Grafton, Auckland 1023, New Zealand.

Melanocytes are melanin-producing cells found in skin, hair follicles, eyes, inner ear, bones, heart and brain of humans. They arise from pluripotent neural crest cells and differentiate in response to a complex network of interacting regulatory pathways. Melanins are pigment molecules that are endogenously synthesized by melanocytes. The light absorption of melanin in skin and hair leads to photoreceptor shielding, thermoregulation, photoprotection, camouflage and display coloring. Melanins are also powerful cation chelators and may act as free radical sinks. Melanin formation is a product of complex biochemical events that starts from amino acid tyrosine and its metabolite, dopa. The types and amounts of melanin produced by melanocytes are determined genetically and are influenced by a variety of extrinsic and intrinsic factors such as hormonal changes, inflammation, age and exposure to UV light. These stimuli affect the different pathways in melanogenesis. In this review we will discuss the regulatory mechanisms involved in melanogenesis and explain how intrinsic and extrinsic factors regulate melanin production. We will also explain the regulatory roles of different proteins involved in melanogenesis.
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http://dx.doi.org/10.3390/ijms17071144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4964517PMC
July 2016

Improving drug retention in liposomes by aging with the aid of glucose.

Int J Pharm 2016 May 25;505(1-2):194-203. Epub 2016 Mar 25.

School of Pharmacy, The University of Auckland, Auckland 1142, New Zealand. Electronic address:

This paper describes a novel method to improve drug retention in liposomes for the poorly water-soluble (lipophilic) model drug asulacrine (ASL). ASL was loaded in the aqueous phase of liposomes and the effects of aging conditions and drug loading levels on drug retention were investigated using an in vitro bio-relevant drug release test established in this study. The status of intra-liposomal drug was investigated using differential scanning calorimetry (DSC) and cryo-transmission electron microscopy (cryo-TEM). Pharmacokinetics and venous tolerance of the formulations were simultaneously studied in rabbits following one-hour intravenous infusion via the ear vein. The presence of glucose during aging was found to be crucial to accelerate drug precipitation and to stabilize the liposomal membrane with high drug loading (8.9% over 4.5% w/w) as a prerequisite. Although no drug crystals were detected, DSC showed a lower phase-transition peak in the glucose-assisted aged ASL-liposomes, indicating interaction of phospholipids with the sugar. Cryo-TEM revealed more 'coffee bean' like drug precipitate in the ASL-liposomes aged in the glucose solution. In rabbits, these liposomes gave rise to a 1.9 times longer half-life than the fresh liposomes, with no venous irritation observed. Inducing and stabilizing drug precipitation in the liposome cores by aging in the presence of sugar provided an easy approach to improve drug retention in liposomes. The study also highlighted the importance of bio-relevance of in vitro release methods to predict in vivo drug release.
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http://dx.doi.org/10.1016/j.ijpharm.2016.03.044DOI Listing
May 2016

Evidence that phospholipase C is involved in the antitumour action of NSC768313, a new thieno[2,3-b]pyridine derivative.

Cancer Cell Int 2016 10;16:18. Epub 2016 Mar 10.

Auckland Cancer Society Research Centre, University of Auckland, Private Bag 92019, Auckland, 1142 New Zealand ; Molecular Medicine and Pathology Department, University of Auckland, Private Bag 92019, Auckland, 1142 New Zealand.

Background: The thieno[2,3-b]pyridines were discovered by virtual high throughput screening as potential inhibitors of phospholipase C (PLC) isoforms and showed potent growth inhibitory effects in National Cancer Institute's human tumour cell line panel (NCI60). The mechanism of the anti-proliferative activity of thieno[2,3-b]pyridines is explored here.

Objectives: We aimed to investigate the basis for the anti-proliferative activity of these thieno[2,3-b]pyridines and to determine whether the cellular inhibition was related to their inhibition of PLC.

Methods: Four breast cancer cell lines were used to assess the anti-proliferative effects (IC50 values) of six representative thieno[2,3-b]pyridines. The most potent compound (derivative 3; NSC768313), was further studied in MDA-MB-231 cells. DNA damage was examined by γH2AX expression level, and cell cycle arrest by flow cytometry. Cell morphology was examined by tubulin antibody staining. The growth inhibitory effect of combination treatment with derivative 3 and paclitaxel (tubulin inhibitor), doxorubicin (topoisomerase II inhibitor) or camptothecin (topoisomerase I inhibitor) was evaluated. A preliminary mouse toxicity assay was used to evaluate the pharmacological properties.

Results: Addition of the thieno[2,3-b]pyridine derivative 3 to the MDA-MB-231 cells induced G2/M growth inhibition, cell cycle arrest in G2-phase, membrane blebbing and the formation of multinucleated cells. It did not induce DNA damage, mitotic arrest or changes in calcium ion flux. Combination of derivative 3 with paclitaxel showed a high degree of synergy, while combinations with doxorubicin and camptothecin showed only additive effects. A mouse pharmacokinetic study of derivative 3 showed that after intraperitoneal injection of a single does (10 mg/Kg), the Cmax was 0.087 μmol/L and the half-life was 4.11 h.

Conclusions: The results are consistent with a mechanism in which thieno[2,3-b]pyridine derivatives interact with PLC isoforms (possibly PLC-δ), which in turn affect the cellular dynamics of tubulin-β, inducing cell cycle arrest in G2-phase. We conclude that these compounds have novelty because of their PLC target and may have utility in combination with mitotic poisons for cancer treatment.
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http://dx.doi.org/10.1186/s12935-016-0293-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4785615PMC
March 2016

The Regulatory Role of Long Noncoding RNAs in Cancer Drug Resistance.

Methods Mol Biol 2016 ;1395:207-27

Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, 85 Park Road, Grafton, Auckland, 1023, New Zealand.

Recent genomic and transcriptomic analysis has revealed that the majority of the human genome is transcribed as nonprotein-coding RNA. These transcripts, known as long noncoding RNA, have structures similar to those of mRNA. Many of these transcripts are now thought to have regulatory roles in different biological pathways which provide cells with an additional layer of regulatory complexity in gene expression and proteome function in response to stimuli. A wide variety of cellular functions may thus depend on the fine-tuning of interactions between noncoding RNAs and other key molecules in cell signaling networks. Deregulation of many noncoding RNAs is thought to occur in a variety of human diseases, including neoplasia and cancer drug resistance. Here we discuss recent findings on the molecular functions of long noncoding RNAs in cellular pathways mediating resistance to anticancer drugs.
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http://dx.doi.org/10.1007/978-1-4939-3347-1_12DOI Listing
November 2016

Classical and Targeted Anticancer Drugs: An Appraisal of Mechanisms of Multidrug Resistance.

Authors:
Bruce C Baguley

Methods Mol Biol 2016 ;1395:19-37

Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, 1142, New Zealand.

The mechanisms by which tumor cells resist the action of multiple anticancer drugs, often with widely different chemical structures, have been pursued for more than 30 years. The identification of P-glycoprotein (P-gp), a drug efflux transporter protein with affinity for multiple therapeutic drugs, provided an important potential mechanism and further work, which identified other members of ATP-binding cassette (ABC) family that act as drug transporters. Several observations, including results of clinical trials with pharmacological inhibitors of P-gp, have suggested that mechanisms other than efflux transporters should be considered as contributors to resistance, and in this review mechanisms of anticancer drug resistance are considered more broadly. Cells in human tumors exist is a state of continuous turnover, allowing ongoing selection and "survival of the fittest." Tumor cells die not only as a consequence of drug therapy but also by apoptosis induced by their microenvironment. Cell death can be mediated by host immune mechanisms and by nonimmune cells acting on so-called death receptors. The tumor cell proliferation rate is also important because it controls tumor regeneration. Resistance to therapy might therefore be considered to arise from a reduction of several distinct cell death mechanisms, as well as from an increased ability to regenerate. This review provides a perspective on these mechanisms, together with brief descriptions of some of the methods that can be used to investigate them in a clinical situation.
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http://dx.doi.org/10.1007/978-1-4939-3347-1_2DOI Listing
November 2016

Potentiation of Growth Inhibitory Responses of the mTOR Inhibitor Everolimus by Dual mTORC1/2 Inhibitors in Cultured Breast Cancer Cell Lines.

PLoS One 2015 6;10(7):e0131400. Epub 2015 Jul 6.

Auckland Cancer Society Research Centre, University of Auckland, Grafton, Auckland, New Zealand.

The mammalian target of rapamycin (mTOR), a vital component of signaling pathways involving PI3K/AKT, is an attractive therapeutic target in breast cancer. Everolimus, an allosteric mTOR inhibitor that inhibits the mTOR functional complex mTORC1, is approved for treatment of estrogen receptor positive (ER+) breast cancer. Other mTOR inhibitors show interesting differences in target specificities: BEZ235 and GSK2126458 are ATP competitive mTOR inhibitors targeting both PI3K and mTORC1/2; AZD8055, AZD2014 and KU-0063794 are ATP competitive mTOR inhibitors targeting both mTORC1 and mTORC2; and GDC-0941 is a pan-PI3K inhibitor. We have addressed the question of whether mTOR inhibitors may be more effective in combination than singly in inhibiting the proliferation of breast cancer cells. We selected a panel of 30 human breast cancer cell lines that included ER and PR positive, HER2 over-expressing, and "triple negative" variants, and determined whether signaling pathway utilization was related to drug-induced inhibition of proliferation. A significant correlation (p = 0.005) was found between everolimus IC50 values and p70S6K phosphorylation, but not with AKT or ERK phosphorylation, consistent with the mTOR pathway being a principal target. We then carried out combination studies with four everolimus resistant triple-negative breast cancer cell lines, and found an unexpectedly high degree of synergy between everolimus and the other inhibitors tested. The level of potentiation of everolimus inhibitory activity (measured by IC50 values) was found to be cell line-specific for all the kinase inhibitors tested. The results suggest that judicious combination of mTOR inhibitors with different modes of action could have beneficial effects in the treatment of breast cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0131400PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4492962PMC
March 2016

Inhibitors of pan-PI3K Signaling Synergize with BRAF or MEK Inhibitors to Prevent BRAF-Mutant Melanoma Cell Growth.

Front Oncol 2015 16;5:135. Epub 2015 Jun 16.

Auckland Cancer Society Research Centre, The University of Auckland , Auckland , New Zealand ; Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland , Auckland , New Zealand.

BRAF and MEK inhibitors have improved outcomes for patients with BRAF-mutant melanoma, but their efficacy is limited by both intrinsic and acquired resistances. Activation of the PI3K pathway can mediate resistance to these agents, providing a strong rationale for combination therapy in melanoma. Here, a panel of nine low-passage human metastatic melanoma cell lines with BRAF mutations was tested in cell proliferation and protein expression assays for sensitivity to inhibitors of MEK (selumetinib) and BRAF (vemurafenib) as single agents and in combination with inhibitors of pan-PI3K (ZSTK474), pan-PI3K/mTOR (BEZ235), individual PI3K isoforms (p110α, A66; p110β, TGX-221; p110γ, AS-252424; p110δ, idelalisib), or mTORC1/2 (KU-0063794). Selumetinib and vemurafenib potently inhibited cell proliferation in all cell lines, especially in those that expressed low levels of phosphorylated AKT (pAKT). ZSTK474 and BEZ235 also inhibited cell proliferation in all cell lines and enhanced the antitumor activity of selumetinib and vemurafenib in the majority of lines by either interacting synergistically or additively to increase potency or by inducing cytotoxicity by significantly increasing the magnitude of cell growth inhibition. Furthermore, ZSTK474 or BEZ235 combined with selumetinib to produce robust inhibition of pERK, pAKT, and pS6 expression and synergistic inhibition of NZM20 tumor growth. The inhibitors of individual PI3K isoforms or mTORC1/2 were less effective at inhibiting cell proliferation either as single agents or in combination with selumetinib or vemurafenib, although KU-0063794 synergistically interacted with vemurafenib and increased the magnitude of cell growth inhibition with selumetinib or vemurafenib in certain cell lines. Overall, these results suggest that the sensitivity of BRAF-mutant melanoma cells to BRAF or MEK inhibitors is at least partly mediated by activation of the PI3K pathway and can be enhanced by combined inhibition of the BRAF/MEK and PI3K/mTOR signaling pathways.
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http://dx.doi.org/10.3389/fonc.2015.00135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4468830PMC
July 2015

Exploring the isoform selectivity of TGX-221 related pyrido[1,2-a]pyrimidinone-based Class IA PI 3-kinase inhibitors: synthesis, biological evaluation and molecular modelling.

Bioorg Med Chem 2015 Jul 4;23(13):3796-808. Epub 2015 Apr 4.

Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand. Electronic address:

A novel series of TGX-221 analogues was prepared and tested for their potency against the p110α, p110β, and p110δ isoforms of the PI3K enzyme, and in two cellular assays. The biological results were interpreted in terms of a p110β comparative model, in order to account for their selectivity towards this isoform. A CH2NH type linker is proposed to allow binding into the specificity pocket proposed to accommodate the high p110β-selectivity of TGX-221, although there was limited steric tolerance for substituents on the pendant ring with the 2-position most favourable for substitution.
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http://dx.doi.org/10.1016/j.bmc.2015.03.073DOI Listing
July 2015

Post-insertion of poloxamer 188 strengthened liposomal membrane and reduced drug irritancy and in vivo precipitation, superior to PEGylation.

J Control Release 2015 Apr 19;203:161-9. Epub 2015 Feb 19.

School of Pharmacy, The University of Auckland, Private Bag 92019, Auckland, New Zealand. Electronic address:

The ultimate aim of this study was to develop asulacrine (ASL)-loaded long-circulating liposomes to prevent phlebitis during intravenous (i.v.) infusion for chemotherapy. Poly(ethylene)glycol (PEG) and poloxamer 188-modified liposomes (ASL-PEGL and ASL-P188L) were developed, and ASL was loaded using a remote loading method facilitated with a low concentration of sulfobutyl ether-β-cyclodextrin as a drug solubilizer. The liposomes were characterized in terms of morphology, size, release properties and stability. Pharmacokinetics and venous tissue tolerance of the formulations were simultaneously studied in rabbits following one-hour i.v. infusion via the ear vein. The irritancy was assessed using a rat paw-lift/lick model after subplantar injections. High drug loading 9.0% w/w was achieved with no drug leakage found from ASL-PEGL or ASL-P188L suspended in a 5% glucose solution at 30days. However, a rapid release (leakage) from ASL-PEGL was observed when PBS was used as release medium, partially related to the use of cyclodextrin in drug loading. Post-insertion of poloxamer 188 to the liposomes appeared to be able to restore the drug retention possibly by increasing the packing density of phospholipids in the membrane. In rabbits (n=5), ASL-P188L had a prolonged half-life with no drug precipitation or inflammation in the rabbit ear vein in contrast to ASL solution. Following subplantar (footpad) injections in rats ASL solution induced paw-lick/lift responses in all rats whereas ASL-P188L caused no response (n=8). PEGylation showed less benefit possibly due to the drug 'leakage'. In conclusion, drug precipitation in the vein and the drug mild irritancy may both contribute to the occurrence of phlebitis caused by the ASL solution, and could both be prevented by encapsulation of the drug in liposomes. Poloxamer 188 appeared to be able to 'seal' the liposomal membrane and enhance drug retention. The study also highlighted the importance of bio-relevant in vitro release study in formulation screening.
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http://dx.doi.org/10.1016/j.jconrel.2015.02.026DOI Listing
April 2015