Pancreatic adenocarcinoma preferentially takes up and is suppressed by synthetic nanoparticles carrying apolipoprotein A-II and a lipid gemcitabine prodrug in mice.

We hypothesised that synthetic HDL nanoparticles carrying a gemcitabine prodrug and apolipoprotein A-II (sHDLGemA2) would target scavenger receptor-B1 (SR-B1) to preferentially and safely deliver gemcitabine into pancreatic ductal adenocarcinoma (PDAC). We designed, manufactured and characterised sHDLGemA2 nanoparticles sized ~130 nm, incorporating 20 mol% of a gemcitabine prodrug within the lipid bilayer, which strengthens on adding ApoA-II. We measured their ability to inhibit growth in cell lines and cell-derived and patient-derived murine PDAC xenografts. Fluorescent-labelled sHDLGemA2 delivered gemcitabine inside xenografts. Xenograft levels of active gemcitabine after sHDLGemA2 were similar to levels after high-dose free gemcitabine. Growth inhibition in mice receiving 4.5 mg gemcitabine/kg/d, carried in sHDLGemA2, was equivalent to inhibition after high-dose (75 mg/kg/d) free gemcitabine, and greater than inhibition after low-dose (4.5 mg/kg/d) free gemcitabine. sHDLGemA2 slowed growth in semi-resistant cells and a resistant human xenograft. sHDLGemA2 targeted xenografts more effectively than sHDLGemA1. SR-B1 was over-expressed in PDAC cells and xenografts. Targeting by ApoA-II was suppressed by anti-SR-B1. Because sHDLGemA2 provided only ~6% of the free gemcitabine dose for an equivalent response, patient side effects can be greatly reduced, and the sHDLGemA2 concept should be developed through clinical trials.

Novel Prognostic Markers in Triple-Negative Breast Cancer Discovered by MALDI-Mass Spectrometry Imaging.

There are no widely-accepted prognostic markers currently available to predict outcomes in patients with triple-negative breast cancer (TNBC), and no targeted therapies with confirmed benefit. We have used MALDI mass spectrometry imaging (MSI) of tryptic peptides to compare regions of cancer and benign tissue in 10 formalin-fixed, paraffin-embedded sections of TNBC tumors. Proteins were identified by reference to a peptide library constructed by LC-MALDI-MS/MS analyses of the same tissues. The prognostic significance of proteins that distinguished between cancer and benign regions was estimated by Kaplan-Meier analysis of their gene expression from public databases. Among peptides that distinguished between cancer and benign tissue in at least 3 tissues with a ROC area under the curve >0.7, 14 represented proteins identified from the reference library, including proteins not previously associated with breast cancer. Initial network analysis using the STRING database showed no obvious functional relationships except among collagen subunits COL1A1, COL1A2, and COL63A, but manual curation, including the addition of EGFR to the analysis, revealed a unique network connecting 10 of the 14 proteins. Kaplan-Meier survival analysis to examine the relationship between tumor expression of genes encoding the 14 proteins, and recurrence-free survival (RFS) in patients with basal-like TNBC showed that, compared to low expression, high expression of nine of the genes was associated with significantly worse RFS, most with hazard ratios >2. In contrast, in estrogen receptor-positive tumors, high expression of these genes showed only low, or no, association with worse RFS. These proteins are proposed as putative markers of RFS in TNBC, and some may also be considered as possible targets for future therapies.

IGFBP-3 interacts with NONO and SFPQ in PARP-dependent DNA damage repair in triple-negative breast cancer.

Women with triple-negative breast cancer (TNBC) are generally treated by chemotherapy but their responsiveness may be blunted by DNA double-strand break (DSB) repair. We previously reported that IGFBP-3 forms nuclear complexes with EGFR and DNA-dependent protein kinase (DNA-PKcs) to modulate DSB repair by non-homologous end-joining (NHEJ) in TNBC cells. To discover IGFBP-3 binding partners involved in chemoresistance through stimulation of DSB repair, we analyzed the IGFBP-3 interactome by LC-MS/MS and confirmed interactions by coimmunoprecipitation and proximity ligation assay. Functional effects were demonstrated by DNA end-joining in vitro and measurement of γH2AX foci. In response to 20 µM etoposide, the DNA/RNA-binding protein, non-POU domain-containing octamer-binding protein (NONO) and its dimerization partner splicing factor, proline/glutamine-rich (SFPQ) formed complexes with IGFBP-3, demonstrated in basal-like TNBC cell lines HCC1806 and MDA-MB-468. NONO binding to IGFBP-3 was also shown in a cell-free biochemical assay. IGFBP-3 complexes with NONO and SFPQ were blocked by inhibiting EGFR with gefitinib or DNA-PKcs with NU7026, and by the PARP inhibitors veliparib and olaparib, which also reduced DNA end-joining activity and delayed the resolution of the γH2AX signal (i.e. inhibited DNA DSB repair). Downregulation of the long noncoding RNA in NHEJ pathway 1 (LINP1) by siRNA also blocked IGFBP-3 interaction with NONO-SFPQ. These findings suggest a PARP-dependent role for NONO and SFPQ in IGFBP-3-dependent DSB repair and the involvement of LINP1 in the complex formation. We propose that targeting of the DNA repair function of IGFBP-3 may enhance chemosensitivity in basal-like TNBC, thus improving patient outcomes.

Proteomic MALDI-TOF/TOF-IMS examination of peptide expression in the formalin fixed brainstem and changes in sudden infant death syndrome infants.

Matrix assisted laser desorption/ionisation imaging mass spectrometry (MALDI-IMS) has not previously been utilised to examine sudden infant death syndrome (SIDS). This study aimed to optimise MALDI IMS for use on archived formalin-fixed-paraffin-embedded human infant medulla tissue (n=6, controls; n=6, SIDS) to evaluate differences between multiple nuclei of the medulla by using high resolution IMS. Profiles were compared between SIDS and age/sex matched controls. LC-MALDI identified 55 proteins based on 321 peptides across all samples; 286 peaks were found using IMS, corresponding to these 55 proteins that were directly compared between controls and SIDS. Control samples were used to identify common peptides for neuronal/non-neuronal structures allowing identification of medullary regions. In SIDS, abnormal expression patterns of 41 peptides (p≤0.05) corresponding to 9 proteins were observed; these changes were confirmed with immunohistochemistry. The protein abnormalities varied amongst nuclei, with the majority of variations in the raphe nuclei, hypoglossal and pyramids. The abnormal proteins are not related to a previously identified neurological disease pathway but consist of developmental neuronal/glial/axonal growth, cell metabolism, cyto-architecture and apoptosis components. This suggests that SIDS infants have abnormal neurological development in the raphe nuclei, hypoglossal and pyramids of the brainstem, which may contribute to the pathogenesis of SIDS. BIOLOGICAL SIGNIFICANCE: This study is the first to perform an imaging mass spectrometry investigation in the human brainstem and also within sudden infant death syndrome (SIDS). LC MALDI and MALDI IMS identified 55 proteins based on 285 peptides in both control and SIDS tissue; with abnormal expression patterns present for 41/285 and 9/55 proteins in SIDS using IMS. The abnormal proteins are critical for neurological development; with the impairment supporting the hypothesis that SIDS may be due to delayed neurological maturation. The brainstem regions mostly affected included the raphe nuclei, hypoglossal and pyramids. This study highlights that basic cyto-architectural proteins are affected in SIDS and that abnormal expression of these proteins in other CNS disorders should be examined. KEY SENTENCES: LC MALDI and MALDI IMS identified 55 proteins based on 285 peptides in both control and SIDS tissue. Abnormal expression patterns were present for 41/285 and 9/55 proteins in SIDS using IMS. Brainstem regions mostly affected included the raphe nuclei, hypoglossal and pyramids.

The effect of ageing on isoniazid pharmacokinetics and hepatotoxicity in Fischer 344 rats.

Isoniazid is the first-line treatment for tuberculosis; however, its use is limited by hepatotoxicity. Age-related differences in isoniazid pharmacokinetics and hepatotoxicity are uncertain. We aimed to investigate these in young (3 ± 0 months, n = 26) and old (23.0 ± 0.2 months, n = 27) male Fischer 344 rats following a low- or high-dose toxic regimen of isoniazid or vehicle (4 doses/day over 2 days; low: 100, 75, 75, 75 mg/kg; high: 150, 105, 105, 105 mg/kg i.p. every 3 h). Fifteen hours after the last dose, animals were euthanized and sera and livers were prepared for analysis. Isoniazid treatment increased serum hepatotoxicity markers (alanine and aspartate transaminase) in young animals but not in old animals, and only reached significance with the high dose in young animals. Isoniazid treatment caused a trend towards an increase in necrosis in young animals with both doses. In contrast, microvesicular steatosis was increased in old isoniazid-treated animals, reaching significance only with the low dose (steatosis prevalence in old: vehicle 1/9, isoniazid 4/5; P < 0.05). Among isoniazid-treated animals, concentrations of toxic intermediates acetylhydrazine and hydrazine were higher in old than young animals (P < 0.05). With both doses, hepatic cytochrome P450 2E1 activity was higher in young animals compared with old (P < 0.05). There were no other age effects seen on any of the other measured enzymes involved in isoniazid metabolism (N-acetyl transferase, amidase, glutathione-S-transferase). These results show age-related changes in isoniazid pharmacokinetics may contribute towards differential patterns of toxicity and confirm that standard hepatotoxicity markers do not detect isoniazid-induced microvesicular steatosis.

A novel truncated form of S100P predicts disease-free survival in patients with lymph node positive breast cancer.

The calcium-binding protein S100P is overexpressed in various cancers and may contribute to the oncogenic phenotype. This study used mass spectrometry to characterize a novel 9.2-kDa C-terminally truncated form of S100P (t-S100P), and to investigate its potential prognostic value in breast cancer. Univariate analysis demonstrated the association between breast tissue t-S100P levels (n = 148) and conventional pathological markers. Across all tumor samples, high t-S100P was strongly prognostic for poor disease-free survival (P = 0.005), its efficacy confined to lymph node-positive tumors (n = 74, P = 0.007). Matrix-assisted laser desorption/ionization imaging mass spectrometry confirmed differential t-S100P abundance between breast cancer and unaffected adjacent tissue. t-S100P was exclusively located in the cell nucleus of breast cancer tissue, and full-length S100P was essentially undetectable by mass spectrometry. We conclude that t-S100P is the predominant form of S100P in breast cancer tissue and is strongly prognostic for disease-free survival in women with lymph node-positive disease.

Novel serum protein biomarker panel revealed by mass spectrometry and its prognostic value in breast cancer.

INTRODUCTION: Serum profiling using proteomic techniques has great potential to detect biomarkers that might improve diagnosis and predict outcome for breast cancer patients (BC). This study used surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS) to identify differentially expressed proteins in sera from BC and healthy volunteers (HV), with the goal of developing a new prognostic biomarker panel. METHODS: Training set serum samples from 99 BC and 51 HV subjects were applied to four adsorptive chip surfaces (anion-exchange, cation-exchange, hydrophobic, and metal affinity) and analyzed by time-of-flight MS. For validation, 100 independent BC serum samples and 70 HV samples were analyzed similarly. Cluster analysis of protein spectra was performed to identify protein patterns related to BC and HV groups. Univariate and multivariate statistical analyses were used to develop a protein panel to distinguish breast cancer sera from healthy sera, and its prognostic potential was evaluated. RESULTS: From 51 protein peaks that were significantly up- or downregulated in BC patients by univariate analysis, binary logistic regression yielded five protein peaks that together classified BC and HV with a receiver operating characteristic (ROC) area-under-the-curve value of 0.961. Validation on an independent patient cohort confirmed the five-protein parameter (ROC value 0.939). The five-protein parameter showed positive association with large tumor size (P = 0.018) and lymph node involvement (P = 0.016). By matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, immunoprecipitation and western blotting the proteins were identified as a fragment of apolipoprotein H (ApoH), ApoCI, complement C3a, transthyretin, and ApoAI. Kaplan-Meier analysis on 181 subjects after median follow-up of >5 years demonstrated that the panel significantly predicted disease-free survival (P = 0.005), its efficacy apparently greater in women with estrogen receptor (ER)-negative tumors (n = 50, P = 0.003) compared to ER-positive (n = 131, P = 0.161), although the influence of ER status needs to be confirmed after longer follow-up. CONCLUSIONS: Protein mass profiling by MS has revealed five serum proteins which, in combination, can distinguish between serum from women with breast cancer and healthy control subjects with high sensitivity and specificity. The five-protein panel significantly predicts recurrence-free survival in women with ER-negative tumors and may have value in the management of these patients.

A DNA-based assay for toxic chemicals in wastewater.

Chemical toxicants, particularly metal ions, are a major contaminant in global waterways. Live-organism bioassays used to monitor chemical toxicants commonly involve measurements of activity or survival of a freshwater cladoceran (Ceriodaphnia dubia) or light emitted by the marine bacterium Vibrio fischeri, used in the commercial Microtox® bioassay. Here we describe a novel molecule-based assay system employing DNA as the chemical biosensor. Metals bind to DNA, causing structural changes that expel a bound (intercalated) fluorescent reporter dye. Analyses of test data using 48 wastewater samples potentially contaminated by metal ions show that the DNA-dye assay results correlate with those from C. dubia and Microtox bioassays. All three assays exhibit additive, antagonistic, and synergistic responses that cannot be predicted by knowing individual metal concentrations. Analyses of metals in these samples imply the presence of chemical toxicants other than metal ions. The DNA-dye assay is robust, has a 12-month shelf life, and is only slightly affected by sample pH in the range 4 to 9. The assay is completed in a matter of minutes, and its portability makes it well suited as a screening assay for use in the field. We conclude that the DNA-dye test is a surrogate bioassay suitable for screening chemical toxicity.

The molecular structure, equilibrium conformation and barrier to internal rotation in decachloroferrocene, Fe(η-C5Cl5)2, determined by gas electron diffraction.

The molecular structure of decachloroferrocene has been determined by gas electron diffraction supported by quantum chemical calculations. The equilibrium conformation has staggered ligand rings and D(5d) symmetry. The barrier to internal rotation is, however, only 0.8(2) kJ mol⁻¹. This barrier is so low that even at room temperature the vast majority of molecules in the gas phase would have sufficient thermal energy to undergo virtually non-hindered internal rotation. While the eclipsed equilibrium conformation of unsubstituted ferrocene is determined by attractive dispersion interaction between the two cyclopentadienyl ligands, the staggered equilibrium conformation of Fe(η-C5Cl5)2 is due to steric repulsion between Cl atoms at different rings. The ligands are non-planar: the C-Cl bonds are bent 3.7(3)° out of the plane of the C5 ring away from the metal atom. The Fe-C, C-C and C-Cl bond distances (r(a)) are: 205.0(4) pm, 143.4(3) pm and 170.2(4) pm respectively.

Sequential treatment of drug-resistant tumors with targeted minicells containing siRNA or a cytotoxic drug.

The dose-limiting toxicity of chemotherapeutics, heterogeneity and drug resistance of cancer cells, and difficulties of targeted delivery to tumors all pose daunting challenges to effective cancer therapy. We report that small interfering RNA (siRNA) duplexes readily penetrate intact bacterially derived minicells previously shown to cause tumor stabilization and regression when packaged with chemotherapeutics. When targeted via antibodies to tumor-cell-surface receptors, minicells can specifically and sequentially deliver to tumor xenografts first siRNAs or short hairpin RNA (shRNA)-encoding plasmids to compromise drug resistance by knocking down a multidrug resistance protein. Subsequent administration of targeted minicells containing cytotoxic drugs eliminate formerly drug-resistant tumors. The two waves of treatment, involving minicells loaded with both types of payload, enable complete survival without toxicity in mice with tumor xenografts, while involving several thousandfold less drug, siRNA and antibody than needed for conventional systemic administration of cancer therapies.

Bacterially-derived nanocells for tumor-targeted delivery of chemotherapeutics and cell cycle inhibitors.

Chemotherapeutic drug therapy in cancer is seriously hampered by severe toxicity primarily due to indiscriminate drug distribution and consequent collateral damage to normal cells. Molecularly targeted drugs such as cell cycle inhibitors are being developed to achieve a higher degree of tumor cell specificity and reduce toxic side effects. Unfortunately, relative to the cytotoxics, many of the molecularly targeted drugs are less potent and the target protein is expressed only at certain stages of the cell cycle thus necessitating regimens like continuous infusion therapy to arrest a significant number of tumor cells in a heterogeneous tumor mass. Here we discuss targeted drug delivery nanovectors and a recently reported bacterially-derived 400 nm sized minicell that can be packaged with therapeutically significant concentrations of chemotherapeutic drugs, targeted to tumor cell surface receptors and effect intracellular drug delivery with highly significant anti-tumor effects in vivo. We also report that molecularly targeted drugs can also be packaged in minicells and targeted to tumor cells with highly significant tumor growth-inhibition and regression in mouse xenografts despite administration of minute amounts of drug. This targeted intracellular drug delivery may overcome many of the hurdles associated with the delivery of cytotoxic and molecularly targeted drugs.

Bacterially derived 400 nm particles for encapsulation and cancer cell targeting of chemotherapeutics.

Systemic administration of chemotherapeutic agents results in indiscriminate drug distribution and severe toxicity. Here we report a technology potentially overcoming these shortcomings through encapsulation and cancer cell-specific targeting of chemotherapeutics in bacterially derived 400 nm minicells. We discovered that minicells can be packaged with therapeutically significant concentrations of chemotherapeutics of differing charge, hydrophobicity, and solubility. Targeting of minicells via bispecific antibodies to receptors on cancer cell membranes results in endocytosis, intracellular degradation, and drug release. This affects highly significant tumor growth inhibition and regression in mouse xenografts and case studies of lymphoma in dogs despite administration of minute amounts of drug and antibody; a factor critical for limiting systemic toxicity that should allow the use of complex regimens of combination chemotherapy.