Professor Christopher Scarlett

Objective: To test the ¿vitamin D-folate hypothesis for the evolution of human skin pigmentation.¿. Methods: Total ozone mapping spectrometer (TOMS) satellite data were used to examine surface UV-irradiance in a large (n¿= 649) Australian cross-sectional study population. Genetic analysis was used to score vitamin D- and folate-related gene polymorphisms (n¿= 22), along with two pigmentation gene variants (IRF4-rs12203592/HERC2-rs12913832). Red cell folate and vitamin D3 were measured by immunoassay and HPLC, respectively. Results: Ultraviolet radiation (UVR) and pigmentation genes interact to modify blood vitamin levels; Light skin IRF4-TT genotype has greatest folate loss while light skin HERC2-GG genotype has greatest vitamin D3 synthesis (reflected in both TOMS and seasonal data). UV-wavelength exhibits a dose¿response relationship in folate loss within light skin IRF4-TT genotype (305 > 310 > 324 > 380 nm). Significant vitamin D3 photosynthesis only occurs within light skin HERC2-GG genotype, and is maximal at 305 nm. Three dietary antioxidants (vitamins C, E, and ß-carotene) interact with UVR and pigmentation genes preventing oxidative loss of labile reduced folate vitamers, with greatest benefit in light skin IRF4-TT subjects. The putative photosensitiser, riboflavin, did not sensitize red cell folate to UVR and actually afforded protection. Four genes (5xSNPs) influenced blood vitamin levels when stratified by pigmentation genotype; MTHFR-rs1801133/rs1801131, TS-rs34489327, CYP24A-rs17216707, and VDR-ApaI-rs7975232. Lightest IRF4-TT/darkest HERC2-AA genotype combination (greatest folate loss/lowest vitamin D3 synthesis) has 0% occurrence. The opposing, commonest (39%) compound genotype (darkest IRF4-CC/lightest HERC2-GG) permits least folate loss and greatest synthesis of vitamin D3. Conclusion: New biophysical evidence supports the vitamin D-folate hypothesis for evolution of skin pigmentation.

Background & Aims: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarker-driven therapeutic strategy targeting DDR and replication stress in PC. Methods: We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient¿derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids. Results: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P <.001) and PARP inhibitor therapy (P <.001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P <.018) and WEE1 inhibitor (P <.029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P <.001) but was not associated with DDR deficiency. Conclusions: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy.

In silico approaches identified 1, N-(6-((4-bromo- benzyl)amino)hexyl)-3,5-bis(trifluoromethyl)benzene sulfonamide, as a potential inhibitor of the S100A2-p53 protein-protein interaction, a validated pancreatic cancer drug target. Subsequent cytotoxicity screening revealed it to be a 2.97 µM cell growth inhibitor of the MiaPaCa-2 pancreatic cell line. This is in keeping with our hypothesis that inhibiting this interaction would have an anti-pancreatic cancer effect with S100A2, the validated PC drug target. A combination of focused library synthesis (three libraries, 24 compounds total) and cytotoxicity screening identified a propyl alkyl diamine spacer as optimal; the nature of the terminal phenyl substituent had limited impact on observed cytotoxicity, whereas N-methylation was detrimental to activity. In total 15 human cancer cell lines were examined, with most analogues showing broad-spectrum activity. Near uniform activity was observed against a panel of six pancreatic cancer cell lines: MiaPaCa-2, BxPC-3, AsPC-1, Capan-2, HPAC and PANC-1. In all cases there was good to excellent correlation between the predicted docking pose in the S100A2-p53 binding groove and the observed cytotoxicity, especially in the pancreatic cancer cell line with high endogenous S100A2 expression. This supports S100A2 as a pancreatic cancer drug target.

In silico screening predicted 1 (N-(4-((4-(3-(4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)propyl)piperazin-1-yl) sulfonyl)-phenyl)acetamide) as an inhibitor of the S100A2-p53 protein-protein interaction. S100A2 is a validated pancreatic cancer drug target. In the MiaPaCa-2 pancreatic cell line, 1 was a ~50 µM growth inhibitor. Synthesis of five focused compound libraries and cytotoxicity screening revealed increased activity from the presence of electron withdrawing moieties on the sulfonamide aromatic ring, with the 3,5-bis-CF3 Library 3 analogues the most active, with GI50 values of 0.91 (3-ClPh; 13 i; BxPC-3, Pancreas) to 9.0 µM (4-CH3; 13 d; PANC-1, Pancreas). Activity was retained against an expanded pancreatic cancer cell line panel (MiaPaCa-2, BxPC-3, AsPC-1, Capan-2, PANC-1 and HPAC) and the normal cell line MCF10A (breast). Bulky 4-disposed substituents on the terminal phenyl ring enhanced broad spectrum activity with growth inhibition values spanning 1.1 to 3.1 µM (4-C(CH3)3; 13 e; BxPC-3 and AsPC-1 (pancreas), respectively). Central alkyl spacer contraction from propyl to ethyl proved detrimental to activity with Library 4 and 5.5- to 10-fold less cytotoxic than the propyl linked Library 2 and Library 3. The data herein was consistent with the predicted binding poses of the compounds evaluated. The highest levels of cytotoxicity were observed with those analogues best capable of adopting a near identical pose to the p53-peptide in the S100A2-p53 binding groove.

Scaevola spinescens is endemic to Australia and traditionally used as a medicinal plant. While its bioactive compounds have been studied, their concentrations in different parts of the plant have not been reported. This study compared total phenolic content (TPC), flavonoids, saponins and antioxidant properties, as well as major individual phytochemical compounds in the whole root, root bark, root wood, whole stem, stem bark, stem wood, and leaf of S. spinescens. The results showed the leaf had significantly highest concentrations of TPC followed by the root bark and stem bark (47.34, 12.24 and 10.20 mg GAE/g, respectively). Flavonoids concentrations were also significantly higher in the leaf compared to the root bark and stem bark (20.95, 6.22 and 4.19 mg CE/g, respectively). For saponins, the root bark contained significantly highest concentrations (112.58 mg EE/g). Luteolin 7-glucoside was isolated and identified in the leaf of S. spinescens. Eight major compounds were identified with the leaf displaying the highest diversity of major compounds, and in higher concentrations, compared to the other plant constituents. As the leaf and root bark contained the highest concentrations of phytochemicals, these plant parts are recommended as starting material for future studies, to further isolate and identify the major compounds from S. spinescens and investigate their biological properties for use in pharmaceutical and food applications.

Scaevola spinescens is endemic to Australia and has traditionally been used by Aboriginal and Torres Strait Islander communities to treat a variety of conditions including colds, flu, fever, stomach pain, urinary disorders, sores, tinea, leprosy, and cancer. Extracts prepared from S. spinescens are non-toxic and have been linked with various medicinal properties including antiviral, antibacterial, anti-inflammatory, and anticancer activities. These studies support the ethnopharmacological use of S. spinescens by Indigenous peoples of Australia and highlight the need for further investigations on the plant for potential use in pharmaceutical and food applications. This review provides a comprehensive, up-to-date review of the literature on S. spinescens focusing on the traditional use, medicinal properties, phytochemicals, and factors that affect their composition during pre-treatment and extraction, as well as providing a framework for future studies of the plant.

The impact of somatic structural variants (SVs) on gene expression in cancer is largely unknown. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole-genome sequencing data and RNA sequencing from a common set of 1220 cancer cases, we report hundreds of genes for which the presence within 100 kb of an SV breakpoint associates with altered expression. For the majority of these genes, expression increases rather than decreases with corresponding breakpoint events. Up-regulated cancer-associated genes impacted by this phenomenon include TERT, MDM2, CDK4, ERBB2, CD274, PDCD1LG2, and IGF2. TERT-associated breakpoints involve ~3% of cases, most frequently in liver biliary, melanoma, sarcoma, stomach, and kidney cancers. SVs associated with up-regulation of PD1 and PDL1 genes involve ~1% of non-amplified cases. For many genes, SVs are significantly associated with increased numbers or greater proximity of enhancer regulatory elements near the gene. DNA methylation near the promoter is often increased with nearby SV breakpoint, which may involve inactivation of repressor elements.

We present SVclone, a computational method for inferring the cancer cell fraction of structural variant (SV) breakpoints from whole-genome sequencing data. SVclone accurately determines the variant allele frequencies of both SV breakends, then simultaneously estimates the cancer cell fraction and SV copy number. We assess performance using in silico mixtures of real samples, at known proportions, created from two clonal metastases from the same patient. We find that SVclone¿s performance is comparable to single-nucleotide variant-based methods, despite having an order of magnitude fewer data points. As part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) consortium, which aggregated whole-genome sequencing data from 2658 cancers across 38 tumour types, we use SVclone to reveal a subset of liver, ovarian and pancreatic cancers with subclonally enriched copy-number neutral rearrangements that show decreased overall survival. SVclone enables improved characterisation of SV intra-tumour heterogeneity.

Cancers require telomere maintenance mechanisms for unlimited replicative potential. They achieve this through TERT activation or alternative telomere lengthening associated with ATRX or DAXX loss. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, we dissect whole-genome sequencing data of over 2500 matched tumor-control samples from 36 different tumor types aggregated within the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium to characterize the genomic footprints of these mechanisms. While the telomere content of tumors with ATRX or DAXX mutations (ATRX/DAXXtrunc) is increased, tumors with TERT modifications show a moderate decrease of telomere content. One quarter of all tumor samples contain somatic integrations of telomeric sequences into non-telomeric DNA. This fraction is increased to 80% prevalence in ATRX/DAXXtrunc tumors, which carry an aberrant telomere variant repeat (TVR) distribution as another genomic marker. The latter feature includes enrichment or depletion of the previously undescribed singleton TVRs TTCGGG and TTTGGG, respectively. Our systematic analysis provides new insight into the recurrent genomic alterations associated with telomere maintenance mechanisms in cancer.

In cancer, the primary tumour¿s organ of origin and histopathology are the strongest determinants of its clinical behaviour, but in 3% of cases a patient presents with a¿metastatic tumour and no obvious primary. Here,as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, we train a deep learning classifier to predict cancer type based on patterns of somatic passenger mutations detected in whole genome sequencing (WGS) of 2606 tumours representing 24 common cancer types produced by the PCAWG Consortium. Our classifier achieves an accuracy of 91% on held-out tumor samples and 88% and 83% respectively on independent primary and metastatic samples, roughly double the accuracy of trained pathologists when presented with a metastatic tumour without knowledge of the primary. Surprisingly, adding information on driver mutations reduced accuracy. Our results have clinical applicability, underscore how patterns of somatic passenger mutations encode the state of the cell of origin, and can inform future strategies to detect the source of circulating tumour DNA.

The type and genomic context of cancer mutations depend on their causes. These causes have been characterized using signatures that represent mutation types that co-occur in the same tumours. However, it remains unclear how mutation processes change during cancer evolution due to the lack of reliable methods to reconstruct evolutionary trajectories of mutational signature activity. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole-genome sequencing data from 2658 cancers across 38 tumour types, we present TrackSig, a new method that reconstructs these trajectories using optimal, joint segmentation and deconvolution of mutation type and allele frequencies from a single tumour sample. In simulations, we find TrackSig has a 3¿5% activity reconstruction error, and 12% false detection rate. It outperforms an aggressive baseline in situations with branching evolution, CNA gain, and neutral mutations. Applied to data from 2658 tumours and 38 cancer types, TrackSig permits pan-cancer insight into evolutionary changes in mutational processes.

Many primary tumours have low levels of molecular oxygen (hypoxia), and hypoxic tumours respond poorly to therapy. Pan-cancer molecular hallmarks of tumour hypoxia remain poorly understood, with limited comprehension of its associations with specific mutational processes, non-coding driver genes and evolutionary features. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2658 cancers across 38 tumour types, we quantify hypoxia in 1188 tumours spanning 27 cancer types. Elevated hypoxia associates with increased mutational load across cancer types, irrespective of underlying mutational class. The proportion of mutations attributed to several mutational signatures of unknown aetiology directly associates with the level of hypoxia, suggesting underlying mutational processes for these signatures. At the gene level, driver mutations in TP53, MYC and PTEN are enriched in hypoxic tumours, and mutations in PTEN interact with hypoxia to direct tumour evolutionary trajectories. Overall, hypoxia plays a critical role in shaping the genomic and evolutionary landscapes of cancer.

Olive pomace is a waste produced by the olive oil industry in massive quantities each year. Disposal of olive pomace is difficult due to high concentrations of phenolic compounds, which is an environmental concern. However, phenolic compounds have applications in the health industry. Therefore, extraction of phenolic compounds from olive pomace has the potential to remove an environmentally hazardous portion of pomace while creating an additional source of income for farmers and producers. Using advanced technologies including Ultrasound Assisted Extraction (UAE), combined with water as an extraction solvent, has recently gained popularity. The present study outlines the optimal UAE conditions for the extraction of phenolic compounds with high antioxidant activity from olive pomace. Optimal conditions were developed using RSM for parameters power, time and sample-to-solvent ratio. Total phenolic compounds determined by Folin Ciocalteu method and total major bioactive compounds determined by HPLC as well as antioxidant capacity (DPPH and CUPRAC) were investigated. The optimal conditions for the extraction of phenolic compounds with high antioxidant activity were 2 g of dried pomace/100 mL of water at 250 W power for 75 min. UAE improved the extraction efficiency of water and yielded extracts with high levels of phenolic compounds and strong antioxidant activity.

Antimicrobial activity of epigallocatechin-3-gallate (EGCG) and two native Australian plants blueberry ash (BBA) fruit and macadamia (MAC) skin extracts against nine pathogenic and spoilage bacteria and seven strains of fungi, using an agar well diffusion assay were investigated. The minimum inhibitory concentrations (MIC) of these compounds were calculated using 96-well microtiter plates method. Finally, active antimicrobial packaging films were prepared by incorporation of EGCG, BBA and MAC extracts at 1-, 2-, 3-, and 4-fold of their correspondence MIC values into edible films based on pea starch and guar gum (PSGG). The antimicrobial activity of films was investigated against target microorganisms by agar disc diffusion technique and quantified using the viable cell count assay. Among the test microorganisms, Salmonella typhimurium and Rhizopus sp. were the most resistance to active films. Films containing EGCG showed the highest activity against all test strains. As the concentration of compounds increased higher than 2¿×¿MIC, the mechanical characteristics of the films were affected considerably. The results indicated that EGCG-PSGG, BBA-PSGG and MAC-PSGG films can be used as active food packaging systems for preserving food safety and prolonging the shelf-life of the packaged food.

The aim of this study was to compare phytochemical and antioxidant properties of different parts of Salacia chinensis L. The root of S. chinensis had the highest content of phenolics, flavonoids, proanthocyanidins and saponins (64.4 mg gallic acid equivalent /g dried weight (DW), 106 mg catechin equivalent (CE)/g DW, 43.5 mg CE/g DW and 799.9 mg escin equivalent /g DW, respectively), followed by the stem and leaf. Similarly, results from the four antioxidant assays (ABTS, DPPH, FRAP and CUPRAC) showed that the root of S. chinensis had the highest antioxidant capacity (632.2, 577.1, 443.3 and 365.0 µM trolox equivalent/g DW, respectively), followed by the stem while the leaf had the lowest antioxidant capacity. From HPLC chromatograms, three major compounds were detected from the root and stem of S. chinensis, while more major individual compounds in lower quantities were seen in the leaf. As the root contained high phytochemical and antioxidant properties, future studies are recommended to isolate and identify the major bioactive compounds from the root for further industrial utilization.

The influence of process variables (pea starch, guar gum and glycerol) on the viscosity (V), solubility (SOL), moisture content (MC), transparency (TR), Hunter parameters (L, a, and b), total color difference (dE), yellowness index (YI), and whiteness index (WI) of the pea starch based edible films was studied using three factors with three level Box-Behnken response surface design. The individual linear effect of pea starch, guar and glycerol was significant (p < 0.05) on all the responses. However, a value was only significantly (p < 0.05) affected by pea starch and guar gum in a positive and negative linear term, respectively. The effect of interaction of starch × glycerol was also significant (p < 0.05) on TR of edible films. Interaction between independent variables starch × guar gum had a significant impact on the b and YI values. The quadratic regression coefficient of pea starch showed a significant effect (p < 0.05) on V, MC, L, b, dE, YI, and WI; glycerol level on dE and WI; and guar gum on dE and SOL value. The results were analyzed by Pareto analysis of variance (ANOVA) and the second order polynomial models were developed from the experimental design with reliable and satisfactory fit with the corresponding experimental data and high coefficient of determination (R2) values (>0.93). Three-dimensional response surface plots were established to investigate the relationship between process variables and the responses. The optimized conditions with the goal of maximizing TR and minimizing SOL, YI and MC were 2.5 g pea starch, 25% glycerol and 0.3 g guar gum. Results revealed that pea starch/guar gum edible films with appropriate physical and optical characteristics can be effectively produced and successfully applied in the food packaging industry.

Integrated genomic analysis of 456 pancreatic ductal adenocarcinomas identified 32 recurrently mutated genes that aggregate into 10 pathways: KRAS, TGF-ß, WNT, NOTCH, ROBO/SLIT signalling, G1/S transition, SWI-SNF, chromatin modification, DNA repair and RNA processing. Expression analysis defined 4 subtypes: (1) squamous; (2) pancreatic progenitor; (3) immunogenic; and (4) aberrantly differentiated endocrine exocrine (ADEX) that correlate with histopathological characteristics. Squamous tumours are enriched for TP53 and KDM6A mutations, upregulation of the TP63¿N transcriptional network, hypermethylation of pancreatic endodermal cell-fate determining genes and have a poor prognosis. Pancreatic progenitor tumours preferentially express genes involved in early pancreatic development (FOXA2/3, PDX1 and MNX1). ADEX tumours displayed upregulation of genes that regulate networks involved in KRAS activation, exocrine (NR5A2 and RBPJL), and endocrine differentiation (NEUROD1 and NKX2-2). Immunogenic tumours contained upregulated immune networks including pathways involved in acquired immune suppression. These data infer differences in the molecular evolution of pancreatic cancer subtypes and identify opportunities for therapeutic development.

Xao tam phan (Paramignya trimera (Oliv.) Guillaum) has been used as an herbal medicine for the treatment of cancer or cancer-like diseases in recent years, particularly in Vietnam. Drying is an important step for preparation of dried materials for storage and further investigation; however, the effects of drying must be taken into account when processing samples, because this can have profound effects on the stability of phytochemical compounds and the biological activity of the dried P. trimera root. As such, this study assessed the effects of four different drying methods (conventional, hot air, vacuum, and microwave) on phytochemical retention and antioxidant capacity of P. trimera root, to identify an optimal drying method for P. trimera root. The results showed that the drying methods significantly affected phytochemical levels and antioxidant capacity of P. trimera root and that among the four drying methods tested, microwave drying (400¿W) had the highest levels of phytochemical compounds, with total phenolic, total flavonoid, proanthocyanidin, and saponin contents of 11.27¿mg GAE, 19.88¿mg RE, 3.98¿mg CE, and 267.15¿mg EE/gram of dried sample, respectively. Dried sample prepared using this method had antioxidant capacity comparable to that of other drying methods. In addition, this method had the shortest drying time (0.28¿h) and consumed the least energy (0.28¿kWh). Therefore, microwave drying should be considered for drying P. trimera root for further investigation and utilization.

The ampulla of Vater is a complex cellular environment from which adenocarcinomas arise to form a group of histopathologically heterogenous tumors. To evaluate the molecular features of these tumors, 98 ampullary adenocarcinomas were evaluated and compared to 44 distal bile duct and 18 duodenal adenocarcinomas. Genomic analyses revealed mutations in the WNT signaling pathway among half of the patients and in all three adenocarcinomas irrespective of their origin and histological morphology. These tumors were characterized by a high frequency of inactivating mutations of ELF3, a high rate of microsatellite instability, and common focal deletions and amplifications, suggesting common attributes in the molecular pathogenesis are at play in these tumors. The high frequency of WNT pathway activating mutation, coupled with small-molecule inhibitors of ß-catenin in clinical trials, suggests future treatment decisions for these patients may be guided by genomic analysis.

The effects of different solvents on the recovery of (i) extractable solids (ES), (ii) total phenolic compounds (TPC), (iii) total flavonoid content (TFC), (iv) vitamin C, and (v) antioxidant activity from lemon pomace waste were investigated. The results revealed that solvents significantly affected the recovery of ES, TPC, TFC, and antioxidant properties. Absolute methanol and 50% acetone resulted in the highest extraction yields of TPC, whereas absolute methanol resulted in the highest extraction of TFC, and water had the highest recovery of vitamin C. 50% ethanol, and 50% acetone had higher extraction yields for TPC, and TFC, as well as higher antioxidant activity compared with their absolute solvents and water. TPC and TFC were shown to be the major components contributing to the antioxidant activity of lemon pomace.

Summary: The papaya (Carica papaya) leaf (PL) contains high levels of saponins and polyphenolic compounds, and historically, it has been used as a folk medicine for numerous ailments, including cancer. PL is traditionally prepared by hot water extraction; however, optimised extraction conditions have not been assessed. This study optimised conditions for the extraction of saponins from PL and assessed their antioxidant capacity and antipancreatic cancer activity. Optimisation was achieved using response surface methodology. Saponins and total phenolic compounds were assessed for their antioxidant, free radical scavenging, ion-reducing capacity, and antipancreatic cancer activity. Optimal aqueous extraction conditions were 85 °C, 25 min. and a water-to-leaf ratio of 20:1 mL g-1. Ethanol extracts demonstrated higher antioxidant, free radical scavenging and ion-reducing capacity, as well as antipancreatic cancer activity. This study revealed that the PL contains numerous bioactive compounds, with significant anticancer activity warranting further studies on the isolation and characterisation of individual bioactive compounds from the PL.

Xao tam phan (. Paramignya trimera (Oliv.) Guillaum) is a Vietnamese traditionally medicinal plant used in the treatment of numerous cancers. The preparation of Xao tam phan extracts including solvent type and extraction method have significant effects on extraction efficiency, phytochemical profile and biological activity. This study aimed to investigate the effects of five various solvents (water, acetonitrile, methanol, ethyl acetate and hexane) and three different extraction methods (conventional, ultrasound-assisted and microwave-assisted) on phytochemical yield and antioxidant capacity of P. trimera root from Vietnam. The results indicate that methanol extracted the maximal yield of phytochemicals from P. trimera and exhibited the greatest antioxidant capacity, with eleven compounds were identified and quantified. Microwave-assisted extraction produced the maximal phytochemical yields (except for total flavonoids) and antioxidant capacity, when compared to conventional and ultrasound-assisted extractions. These data reveal that the use of methanol and microwave-assisted extraction are recommended for extraction of biologically active phytochemicals from P. trimera root for application in the nutraceutical and/or pharmaceutical industries.

Ovarian cancer is the most common cause of death among women with gynecologic cancer. We examined molecular profiles of fibroblasts from normal ovary and high-grade serous ovarian tumors to identify novel therapeutic targets involved in tumor progression. We identified 2,300 genes that are significantly differentially expressed in tumor-associated fibroblasts. Fibroblast expression of one of these genes, connective tissue growth factor (CTGF), was confirmed by immunohistochemistry. CTGF protein expression in ovarian tumor fibroblasts significantly correlated with gene expression levels. CTGF is a secreted component of the tumor microenvironment and is being pursued as a therapeutic target in pancreatic cancer. We examined its effect in in vitro and ex vivo ovarian cancer models, and examined associations between CTGF expression and clinico-pathologic characteristics in patients. CTGF promotes migration and peritoneal adhesion of ovarian cancer cells. These effects are abrogated by FG-3019, a human monoclonal antibody against CTGF, currently under clinical investigation as a therapeutic agent. Immunohistochemical analyses of high-grade serous ovarian tumors reveal that the highest level of tumor stromal CTGF expression was correlated with the poorest prognosis. Our findings identify CTGF as a promoter of peritoneal adhesion, likely to mediate metastasis, and a potential therapeutic target in high-grade serous ovarian cancer. These results warrant further studies into the therapeutic efficacy of FG-3019 in high-grade serous ovarian cancer.

Despite incremental advances in the diagnosis and treatment for pancreatic cancer (PC), the 5-year survival rate remains <5%. Novel therapies to increase survival and quality of life for PC patients are desperately needed. Epigenetic therapeutic agents such as histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) have demonstrated therapeutic benefits in human cancer. We assessed the efficacy of these epigenetic therapeutic agents as potential therapies for PC using in vitro and in vivo models. Treatment with HDACi [suberoylanilide hydroxamic acid (SAHA)] and DNMTi [5-AZA-2' deoxycytidine (5-AZA-dc)] decreased cell proliferation in MiaPaCa2 cells, and SAHA treatment, with or without 5-AZA-dc, resulted in higher cell death and lower DNA synthesis compared to 5-AZA-dc alone and controls (DMSO). Further, combination treatment with SAHA and 5-AZA-dc significantly increased expression of p21WAF1, leading to G1 arrest. Treatment with epigenetic agents delayed tumour growth in vivo, but did not decrease growth of established pancreatic tumours. In conclusion, these data demonstrate a potential role for epigenetic modifier drugs for the management of PC, specifically in the chemoprevention of PC, in combination with other chemotherapeutic agents.

Far-infrared (FIR) vacuum is an advanced drying technique that has recently been applied in food processing. Optimal drying conditions for processing tea from Miang leaves using FIR vacuum drying were investigated. Response surface methodology with a central composite design was used to design, analyze, and predict the optimal time and temperature conditions for FIR vacuum drying, taking into account the physicochemical properties of Miang leaves. When the temperature increased from 50 to 65°C and the time from 60 to 120 min, the amount of epicatechin, epicatechin gallate, epigallocatechin gallate, and total catechins significantly (p<0.05) increased while the moisture content and water activity significantly (p<0.05) decreased, compared with controls. The physicochemical properties of dried Miang leaves were significantly (p>0.05) influenced by time and temperature, compared with controls. Drying conditions of 65°C for 120 min are recommended for optimization of drying.

Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.

© 2015 Wiley Periodicals, Inc. Vitex agnus-castus (VitexAC) leaves have been used for medicinal purposes for many years. Drying is important to prepare starting materials for further processing, as it is associated with production cost and quality of the materials. Herein, the effects of five different drying conditions on the physical, chemical and antioxidant properties of VitexAC were evaluated. The results showed that 30% of dried leaves with moisture content of less than 7% could be produced from fresh leaves. VitexAC leaves dried by freeze and vacuum drying at 65C had higher levels of bioactive compounds as well as higher antioxidant capacity in comparison with other drying conditions, revealing that these drying conditions are more suitable for drying VitexAC leaves. However, freeze drying is costly and time-consuming; thus, vacuum drying at 65C is suggested for drying the VitexAC leaves as the starting materials for further processing steps. Practical Applications: As drying is an important process to prepare starting materials for further processing steps, it is important to compare different drying methods to identify the most suitable method with low cost and less effect on material quality. The results showed that inappropriate drying conditions resulted in big loss of bioactive compounds and antioxidant capacity. Vacuum drying at 65C was found to be the most suitable method, which can be easily applied for drying Vitex agnus-castus leaves in the industrial scale.

The genus Eucalyptus (Myrtaceae) is mainly native to Australia; however, some species are now distributed globally. Eucalyptus has been used in indigenous Australian medicines for the treatment of a range of aliments including colds, flu, fever, muscular aches, sores, internal pains, and inflammation. Eucalyptus oils containing volatile compounds have been widely used in the pharmaceutical and cosmetics industries for a multitude of purposes. In addition, Eucalyptus extracts containing nonvolatile compounds are also an important source of key bioactive compounds, and several studies have linked Eucalyptus extracts with anticancer properties. With the increasing research interest in Eucalyptus and its health properties, this review briefly outlines the botanical features of Eucalyptus, discusses its traditional use as medicine, and comprehensively reviews its phytochemical and anticancer properties and, finally, proposes trends for future studies.

Myc oncoproteins and histone deacetylases (HDACs) exert oncogenic effects by modulating gene transcription. Paradoxically, N-Myc induces p53 gene expression. Tumor protein 53-induced nuclear protein 1 (TP53INP1) phosphorylates p53 protein at serine 46, leading to enhanced p53 activity, transcriptional activation of p53 target genes and programmed cell death. Here we aimed to identify the mechanism through which N-Myc overexpressing p53 wild-type neuroblastoma cells acquired resistance to apoptosis. TP53INP1 was found to be one of the genes most significantly repressed by HDAC2 and N-Myc according to Affymetrix microarray gene expression datasets. HDAC2 and N-Myc reduced TP53INP1 gene expression by direct binding to the TP53INP1 gene promoter, leading to transcriptional repression of TP53INP1, p53 protein de-phosphorylation at serine 46, neuroblastoma cell proliferation and survival. Moreover, low levels of TP53INP1 expression in human neuroblastoma tissues correlated with high levels of N-Myc expression and poor patient outcome, and the BET bromodomain inhibitors JQ1 and I-BET151 reduced N-Myc expression and reactivated TP53INP1 expression in neuroblastoma cells. These findings identify TP53INP1 repression as an important co-factor for N-Myc oncogenesis, and provide further evidence for the potential application of BET bromodomain inhibitors in the therapy of N-Myc-induced neuroblastoma.

Background:Adjuvant chemotherapy improves survival for patients with resected pancreatic cancer. Elderly patients are under-represented in Phase III clinical trials, and as a consequence the efficacy of adjuvant therapy in older patients with pancreatic cancer is not clear. We aimed to assess the use and efficacy of adjuvant chemotherapy in older patients with pancreatic cancer.Methods:We assessed a community cohort of 439 patients with a diagnosis of pancreatic ductal adenocarcinoma who underwent operative resection in centres associated with the AUSn Pancreatic Cancer Genome Initiative.Results:The median age of the cohort was 67 years. Overall only 47% of all patients received adjuvant therapy. Patients who received adjuvant chemotherapy were predominantly younger, had later stage disease, more lymph node involvement and more evidence of perineural invasion than the group that did not receive adjuvant treatment. Overall, adjuvant chemotherapy was associated with prolonged survival (median 22.1 vs 15.8 months; P<0.0001). Older patients (aged =70) were less likely to receive adjuvant chemotherapy (51.5% vs 29.8%; P<0.0001). Older patients had a particularly poor outcome when adjuvant therapy was not delivered (median survival=13.1 months; HR 1.89, 95% CI: 1.27-2.78, P=0.002).Conclusion:Patients aged =70 are less likely to receive adjuvant therapy although it is associated with improved outcome. Increased use of adjuvant therapy in older individuals is encouraged as they constitute a large proportion of patients with pancreatic cancer. © 2014 Cancer Research UK.

Objective: Pancreatic cancer is a leading cause of cancer-related death in the Western world. Current chemotherapy regimens have modest survival benefit. Thus, novel, effective therapies are required for treatment of this disease. Design Activating KRAS mutation almost always drives pancreatic tumour initiation, however, deregulation of other potentially druggable pathways promotes tumour progression. PTEN loss leads to acceleration of Kras G12Ddriven pancreatic ductal adenocarcinoma (PDAC) in mice and these tumours have high levels of mammalian target of rapamycin (mTOR) signalling. To test whether these KRAS PTEN pancreatic tumours show mTOR dependence, we compared response to mTOR inhibition in this model, to the response in another established model of pancreatic cancer, KRAS P53. We also assessed whether there was a subset of pancreatic cancer patients who may respond to mTOR inhibition. Results: We found that tumours in KRAS PTEN mice exhibit a remarkable dependence on mTOR signalling. In these tumours, mTOR inhibition leads to proliferative arrest and even tumour regression. Further, we could measure response using clinically applicable positron emission tomography imaging. Importantly, pancreatic tumours driven by activated KRAS and mutant p53 did not respond to treatment. In human tumours, approximately 20% of cases demonstrated low PTEN expression and a gene expression signature that overlaps with murine KRAS PTEN tumours. Conclusions: KRAS PTEN tumours are uniquely responsive to mTOR inhibition. Targeted anti-mTOR therapies may offer clinical benefit in subsets of human PDAC selected based on genotype, that are dependent on mTOR signalling. Thus, the genetic signatures of human tumours could be used to direct pancreatic cancer treatment in the future.

Lilly pilly (LP) fruit (Syzygium paniculatum Gaertn.) is widely grown in eastern Australia and has been used as food by indigenous Australians. However, there is limited information on its bioactivity. This study investigated the physicochemical and antioxidant properties of the crude fruit extract, identified its bioactive compounds and also assessed its potential anti-proliferative effect on pancreatic cancer cells. Our data showed that the LP extract was water-soluble and possessed a total phenolic content of 96 mg of gallic acid equivalents (GAE)/g, flavonoid levels of 52 mg catechin equivalents (CAE)/g, proanthocyanidin levels of 29 mg CAE/g. Several phenolic compounds such as gallic acid, chlorogenic acid, catechin and epicatechin were identified in the LP extract with levels of 0.39, 2.35, 0.47 and 2.9 mg/g, respectively. Results from six different antioxidant assays revealed that the LP extract pocessed potent antioxidant and free radical scavenging capacity. Although antioxidant capacity of the extract was lower than that of vitamin E, vitamin C and BHT, it could be significantly improved if the extract was to be further purified. We also showed that the LP extract (200 µg/mL) significantly reduced the viability of MiaPaCa-2 and ASPC-1 pancreatic cancer cells to levels comparable to that of the chemotherapeutic agent gemcitabine. For this reason lilly pilly should be further investigated for its health promoting and potential anti-cancer benefits, particularly for pancreatic cancer. © 2014 Elsevier GmbH.

The N-Myc oncoprotein induces neuroblastoma, which arises from undifferentiated neuroblasts in the sympathetic nervous system, by modulating gene and protein expression and consequently causing cell differentiation block and cell proliferation. The class IIa histone deacetylase 5 (HDAC5) represses gene transcription, and blocks myoblast, osteoblast and leukemia cell differentiation. Here we showed that N-Myc upregulated HDAC5 expression in neuroblastoma cells. Conversely, HDAC5 repressed the ubiquitin-protein ligase NEDD4 gene expression, increased Aurora A gene expression and consequently upregulated N-Myc protein expression. Genome-wide gene expression analysis and protein co-immunoprecipitation assays revealed that HDAC5 and N-Myc repressed the expression of a common subset of genes by forming a protein complex, whereas HDAC5 and the class III HDAC SIRT2 independently repressed the expression of another common subset of genes without forming a protein complex. Moreover, HDAC5 blocked differentiation and induced proliferation in neuroblastoma cells. Taken together, our data identify HDAC5 as a novel co-factor in N-Myc oncogenesis, and provide the evidence for the potential application of HDAC5 inhibitors in the therapy of N-Myc-induced neuroblastoma and potentially other c-Myc-induced malignancies.. © 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14.

Background: The diagnosis of pancreatic cystic lesions is problematical with difficulties arising in the differentiation between malignant, premalignant or benign lesions. This preliminary study aimed to analyse pancreatic cyst fluid, using a proteomic approach, to generate reproducible protein profiles to assist in the classification of malignant and non-carcinoma samples. Methods: Pancreatic cyst fluid samples from patients with pancreatic adenocarcinoma and non-carcinoma cystic lesions were analysed on hydrophobic protein chip arrays by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Results: Differential protein expression profiles were observed between pancreatic adenocarcinoma and non-carcinoma cyst fluid samples using SELDI-TOF MS, with 12 protein peaks differentially expressed between pancreatic adenocarcinoma and non-carcinoma. Additionally, unique patterns were observed between the different subtypes of non-carcinoma samples as well as malignant adenocarcinoma. Conclusions: In this preliminary study we used SELDI-TOF MS to identify protein expression profiles of pancreatic cyst fluid, showing a potential to aid in the differential diagnosis of pancreatic cystic lesions. © 2007 Royal Australasian College of Surgeons.

Background & Aims: Pancreatic adenocarcinoma is a most devastating cancer that presents late and is rapidly progressive. This study aimed to identify unique, tissue-specific protein biomarkers capable of differentiating pancreatic adenocarcinoma (PC) from adjacent uninvolved pancreatic tissue (AP), benign pancreatic disease (B), and nonmalignant tumor tissue (NM). Methods: Tissue samples representing PC (n = 31), AP (n = 44), and B (n = 19) tissue were analyzed on hydrophobic protein chip arrays by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Training models were developed using logistic regression and validated using the 10-fold cross-validation approach. Results: The hydrophobic protein chip array revealed 13 protein peaks differentially expressed between PC and AP (receiver operating characteristic [ROC] area under the curve [AUC], 0.64-0.85), 8 between PC and B (ROC AUC, 0.67-0.78), and 12 between PC and NM tissue (ROC AUC, 0.63-0.81). Logistic regression and cross-validation identified overlapping panels of peaks to develop a training model that distinguished PC from AP (77.4% sensitivity, 84.1% specificity), B (83.9% sensitivity, 78.9% specificity), and NM tissue (58.1% sensitivity, 90.5% specificity). The final panels selected correctly classified 80.6% of PC and 88.6% of AP samples (ROC AUC, 0.92), 93.5% of PC and 89.5% of B samples (ROC AUC, 0.99), and 71.0% of PC and 92.1% of NM samples (ROC AUC, 0.91). Conclusions: This study used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to discover a number of protein panels that can distinguish effectively between pancreatic adenocarcinoma, benign, and adjacent pancreatic tissue. Identification of these proteins will add to our understanding of the biology of pancreatic cancer. Furthermore, these protein panels may have important diagnostic implications. © 2006 American Gastroenterological Association Institute.

Proteomic techniques promise to improve the diagnosis of cholangiocarcinoma (CC) in both tissue and serum as histological diagnosis and existing serum markers exhibit poor sensitivities. We explored the use of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify potential protein biomarkers of CC. Twenty-two resected CC samples were compared with adjacent noninvolved bile duct tissue. Serum from patients with CC (n = 20) was compared with patients with benign disease (n = 20), and healthy volunteers (n = 25). Samples were analyzed on hydrophobic protein chips via SELDI-TOF MS, and classification models were developed using logistic regression and cross-validation analysis. Univariate analysis revealed 14 individual peaks differentially expressed between CC and bile duct tissue, 4 peaks between CC and benign disease, and 12 peaks between CC and sera of healthy volunteers. The 4,462 mass-to-charge serum peak had superior discriminatory ability to carbohydrate antigen 19.9 (CA19.9) and carcinoembryonic antigen (CEA) (P = .004; receiver operating characteristic [ROC] area under the curve [AUC] = 0.76, 0.73, and 0.70, respectively). The training models developed panels of peaks that distinguished CC from bile duct tissue (92.5% sensitivity, 92.3% specificity, ROC AUC = 0.96), CC from benign serum (65.0% sensitivity, 70.0% specificity, ROC AUC = 0.83), and CC from sera of healthy volunteers (75.0% sensitivity, 100% specificity; ROC AUC = 0.92). Serum results were further improved with the inclusion of CA19.9 and CEA (ROC AUC = 0.86 and 0.99 for CC vs benign and healthy volunteer serum, respectively). In conclusion, biomarker panels are capable of distinguishing CC from nonmalignant tissue; serum markers have important diagnostic implications for unknown bile duct stricture. Copyright © 2006 by the American Association for the Study of Liver Diseases.

HER-2 is a transmembrane growth factor receptor recognized in overexpression as an independent adverse prognostic factor in several cancers. This study measured HER-2 overexpression in pancreatic adenocarcinoma at the genetic, transcriptional, and translational level. Expression was gauged with regard to stage, grade, and survival. Pancreatic adenocarcinoma samples (n = 30) were analyzed with immunohistochemical labeling for HER-2 protein, Quantitative real-time reverse transcriptase polymerase chain reaction (Q-RT-PCR) measurement of HER-2 mRNA and fluorescence in situ hybridization (FISH) analysis of HER-2 gene expression. HER-2 expression in benign pancreatic lesions (n = 10) provided a control. Five (17%) of the pancreatic adenocarcinomas scored maximal 3+ immunohistochemistry (IHC) labeling, seven (23%) had significantly increased expression of HER-2 mRNA, while only one (3%) exhibited low level HER-2 gene amplification. Ten (33%) tumors demonstrated aneuploidy. In general, concordance between methodologies was poor, but the best agreement was seen between FISH aneuploidy status and Q-RT-PCR mRNA overexpression (80% agreement), followed by IHC and Q-RT-PCR (73% agreement). The least agreement was seen between IHC and FISH aneuploidy status (67% agreement). Tumor stage was positively associated with HER-2 mRNA and protein expression, but tumor grade and other patient characteristics did not reach statistical significance. A poor survival outcome was demonstrated with positive HER-2 status in all three measures of overexpression (Kaplan-Meier log-rank score; P < 0.01 [IHC], P = 0.05 [Q-RT-PCR], P = 0.02 [FISH]). Discordance in expression at the nuclear, cytoplasmic, and cell surface levels highlights the limitations of immunohistochemical evaluation alone and stresses the need for further evaluation of response to anti-HER-2 targeted therapies in tumors displaying overexpression in gene copy, mRNA, and receptor protein. Copyright © 2005 by Lippincott Williams & Wilkins.

Background and Aims: Overexpression of urokinase-type plasminogen activator (uPA) has been shown to be strongly associated with an increased metastatic potential and poor prognosis in a variety of human malignancies. It was hypothesized that uPA would be overexpressed in highly metastatic pancreatic cancer. The aims of this study were to analyze uPA mRNA expression in pancreatic cancer and to correlate this to the expression of uPA protein and to the stage of the disease. Methods: Twenty-one pancreatic adenocarcinoma, six ampullary carcinoma and 10 benign mucinous cystadenoma samples, all with adjacent normal tissue, were collected. uPA mRNA was measured using real-time quantitative reverse transcription polymerase chain reaction. Localization of uPA within normal and pancreatic tumor sections was subsequently confirmed using immunohistochemistry. Results: The median and range of the ratios of uPA mRNA measures between tumor tissue and non-involved pancreatic tissue was 17.1 (1.4-653.6) for pancreatic adenocarcinoma (P < 0.001), 3.9 (0.7-7.7) for ampullary carcinoma (P = 0.055) and 1.9 (0.6-5.9) for mucinous cystadenoma tissue (P = 0.052). uPA low tumors were associated with an exuberant stromal reaction, whereas uPA high tumors showed little stromal response. Immunohistochemistry confirmed that uPA protein was more prevalent in pancreatic adenocarcinoma tissue than in normal tissue and that it was membrane-bound. uPA mRNA expression was significantly associated with poorly differentiated pancreatic cancers (P < 0.05) and positively associated with tumor stage. Conclusions: These observations suggest that significant overexpression of uPA correlates closely to the rapid progression and invasiveness of pancreatic cancer and that uPA may provide a future therapeutic target for pancreatic cancer treatment. © 2004 Blackwell Publishing Asia Pty Ltd.

Background & aims: Methods of nutritional management in abdominal sepsis remain controversial. Methods: Sprague Dawley rats were either fed via a central line in the right internal jugular vein or duodenally via a gastrostomy tube, and were randomised to undergo either caecal ligation and puncture (CLP) or laparotomy only. Post-operatively, animals received either parenteral nutrition, enteral nutrition or saline only (parenteral and enteral nutrition protocols were isocaloric and isonitrogenous). After 72 h, fractional rate of protein synthesis (Ks, %/day) was measured in gastrocnemius muscle and liver, and protein breakdown was measured in incubated epitrochlearis muscles. Serum insulin-like growth factor-I (IGF-I), acid-labile subunit (ALS) and IGF binding protein-1 (IGFBP-1) levels were determined by specific radioimmunoassay methods. Results: After CLP, when compared with starved animals, only enteral nutrition resulted in a significant decrease in survival to 7 h (P<0.001). Parenteral nutrition, but not enteral nutrition, increased muscle (P=0.02) and liver (P<0.001) Ks, IGF-I (P<0.001) and ALS levels (P<0.001), whereas both parenteral and enteral nutrition reduced IGFBP-1 levels (P<0.001). Neither enteral nor parenteral nutrition reduced protein breakdown in septic animals. Conclusions: In this model of severe abdominal sepsis where gut function cannot be assessed, enteral nutrition was associated with increased mortality and was less effective than parenteral nutrition in augmenting muscle and liver protein synthesis. © 2004 Elsevier Ltd. All rights reserved.