Danielle R. Bond

Exploring the least studied Australian eucalypt genera: Corymbia and Angophora for phytochemicals with anticancer activity against pancreatic malignancies.

While the pharmacological and toxicological properties of eucalypts are well known in indigenous Australian medicinal practice, investigations of the bioactivity of eucalypt extracts against high mortality diseases such as pancreatic cancer in Western medicine have to date been limited, particularly amongst the genera Corymbia and Angophora. Four Angophora and Corymbia species were evaluated for their phytochemical profile and efficacy against both primary and secondary pancreatic cancer cell lines. The aqueous leaf extract of Angophora hispida exhibited statistically higher total phenolic content (107.85 ± 1.46 mg of gallic acid equiv. per g) and total flavonoid content (57.96 ± 1.93 mg rutin equiv. per g) and antioxidant capacity compared to the other tested eucalypts (P < 0.05). Both A. hispida and A. floribunda aqueous extracts showed statistically similar saponin contents. Angophora floribunda extract exerted significantly greater cell growth inhibition of 77.91 ± 4.93% followed by A. hispida with 62.04 ± 7.47% (P < 0.05) at 100 μg/ml in MIA PaCa‐2 cells with IC50 values of 75.58 and 87.28 μg/ml, respectively. More studies are required to isolate and identify the bioactive compounds from these two Angophora species and to determine their mode of action against pancreatic malignancies.

Extracellular vesicles with altered tetraspanin CD9 and CD151 levels confer increased prostate cell motility and invasion

To facilitate intercellular communication, cells release nano-sized, extracellular vesicles (EVs) to transfer biological cargo to both local and distant sites. EVs are enriched in tetraspanins, two of which (CD9 and CD151) have altered expression patterns in many solid tumours, including prostate cancer, as they advance toward metastasis. We aimed to determine whether EVs from prostate cells with altered CD9 and CD151 expression could influence cellular behaviour and increase the metastatic capabilities of non-tumourigenic prostate cells. EVs were isolated by ultrafiltration and characterised for their tetraspanin expression and size distribution. iTRAQ was used to identify differences between RWPE1 and tetraspanin-modified RWPE1 EV proteomes, showing an enrichment in protein degradation pathways. Addition of EVs from RWPE1 cells with reduced CD9 or increased CD151 abundance resulted in increased invasion of RWPE1 cells, and increased migration in the case of high CD151 abundance. We have been able to show that alteration of CD9 and CD151 on prostate cells alters the proteome of their resultant EVs, and that these EVs can enhance the migratory and invasive capabilities of a non-tumourigenic prostate cellular population. This work suggests that cellular tetraspanin levels can alter EVs, potentially acting as a driver of metastasis in prostate cancer.

Animal models of pancreatic cancer and their application in clinical research

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Lipidomic profiling of extracellular vesicles derived from prostate and prostate cancer cell lines

BackgroundExtracellular vesicles (EVs) are produced and secreted from most cells of the body and can be recovered in biological fluids. Although there has been extensive characterisation of the protein and nucleic acid component of EVs, their lipidome has received little attention and may represent a unique and untapped source of biomarkers for prostate cancer diagnosis and prognosis.MethodsEVs were isolated from non-tumourigenic (RWPE1), tumourigenic (NB26) and metastatic (PC-3) prostate cell lines. Lipids were extracted and subsequently used for targeted lipidomics analysis for the quantitation of molecular lipid species.ResultsA total of 187 molecular lipid species were quantitatively identified in EV samples showing differential abundance between RWPE1, NB26 and PC-3 EV samples. Fatty acids, glycerolipids and prenol lipids were more highly abundant in EVs from non-tumourigenic cells, whereas sterol lipids, sphingolipids and glycerophospholipids were more highly abundant in EVs from tumourigenic or metastatic cells.ConclusionsThis study identified differences in the molecular lipid species of prostate cell-derived EVs, increasing our understanding of the changes that occur to the EV lipidome during prostate cancer progression. These differences highlight the importance of characterising the EV lipidome, which may lead to improved diagnostic and prognostic biomarkers for prostate cancer.

The olive biophenols oleuropein and hydroxytyrosol selectively reduce proliferation, influence the cell cycle, and induce apoptosis in pancreatic cancer cells

Current chemotherapy drugs for pancreatic cancer only offer an increase in survival of up to six months. Additionally, they are highly toxic to normal tissues, drastically affecting the quality of life of patients. Therefore, the search for novel agents, which induce apoptosis in cancer cells while displaying limited toxicity towards normal cells, is paramount. The olive biophenols, oleuropein, hydroxytyrosol and tyrosol, have displayed cytotoxicity towards cancer cells without affecting non-tumorigenic cells in cancers of the breast and prostate. However, their activity in pancreatic cancer has not been investigated. Therefore, the aim of this study was to determine the anti-pancreatic cancer potential of oleuropein, hydroxytyrosol and tyrosol. Pancreatic cancer cells (MIA PaCa-2, BxPC-3, and CFPAC-1) and non-tumorigenic pancreas cells (HPDE) were treated with oleuropein, hydroxytyrosol and tyrosol to determine their effect on cell viability. Oleuropein displayed selective toxicity towards MIA PaCa-2 cells and hydroxytyrosol towards MIA PaCa-2 and HPDE cells. Subsequent analysis of Bcl-2 family proteins and caspase 3/7 activation determined that oleuropein and hydroxytyrosol induced apoptosis in MIA PaCa-2 cells, while oleuropein displayed a protective effect on HPDE cells. Gene expression analysis revealed putative mechanisms of action, which suggested that c-Jun and c-Fos are involved in oleuropein and hydroxytyrosol induced apoptosis of MIA PaCa-2 cells.

Eucalyptus microcorys leaf extract derived HPLC-fraction reduces the viability of MIA PaCa-2 cells by inducing apoptosis and arresting cell cycle

New therapeutic strategies such as the development of novel drugs and combinatorial therapies with existing chemotherapeutic agents are urgently needed to improve the clinical prognosis of pancreatic cancer. We have previously reported the antiproliferative properties of aqueous crude Eucalyptus microcorys extract against pancreatic cancer cell lines. In this study, bioassay-guided fractionation of the aqueous crude E. microcorys extract using RP-HPLC and subsequent assessment of the resultant fractions (F1-F5) for their antioxidant activity and cytotoxicity against pancreatic cancer cell lines were performed. The molecular mechanisms associated with the cytotoxicity was characterised by studying the effects of the most potent fraction-1 (F1) on apoptosis and cell cycle profiles as well as its phytochemical constituents by LC-ESI/MS/MS. F1 displayed significantly greater antioxidant activity in three different assays (p < 0.05). Moreover, F1 exhibited significantly greater antiproliferative activity (IC50 = 93.11 ± 3.43 μg/mL) against MIA PaCa-2 cells compared to the other four fractions (p < 0.05). F1 induced apoptosis by regulating key apoptotic proteins- Bcl-2, Bak, Bax, cleaved PARP, procaspase-3 and cleaved caspase-3 in MIA PaCa-2 cells, suggesting the involvement of intrinsic mitochondrial apoptotic pathway and arrested cells at G2/M phase. A combination of gemcitabine and F1 exerted a greater effect on apoptosis and cell cycle arrest than F1 or gemcitabine alone (p < 0.05). LC-ESI/MS/MS revealed the tentative identities of phytochemicals present in F1 and their similarities with the phenolic compounds previously reported in Eucalyptus with antipancreatic cancer activity. Our study shows that the polyphenol and antioxidant-rich fraction of E. microcorys extract is a promising candidate for developing mono or combination therapies against pancreatic cancer.

Characterization of the early molecular changes in the glomeruli of Cd151 −/− mice highlights induction of mindin and MMP-10

In humans and FVB/N mice, loss of functional tetraspanin CD151 is associated with glomerular disease characterised by early onset proteinuria and ultrastructural thickening and splitting of the glomerular basement membrane (GBM). To gain insight into the molecular mechanisms associated with disease development, we characterised the glomerular gene expression profile at an early stage of disease progression in FVB/N Cd151−/− mice compared to Cd151+/+ controls. This study identified 72 up-regulated and 183 down-regulated genes in FVB/N Cd151−/− compared to Cd151+/+ glomeruli (p < 0.05). Further analysis highlighted induction of the matrix metalloprotease MMP-10 and the extracellular matrix protein mindin (encoded by Spon2) in the diseased FVB/N Cd151−/− GBM that did not occur in the C57BL/6 diseased-resistant strain. Interestingly, mindin was also detected in urinary samples of FVB/N Cd151−/− mice, underlining its potential value as a biomarker for glomerular diseases associated with GBM alterations. Gene set enrichment and pathway analysis of the microarray dataset showed enrichment in axon guidance and actin cytoskeleton signalling pathways as well as activation of inflammatory pathways. Given the known function of mindin, its early expression in the diseased GBM could represent a trigger of both further podocyte cytoskeletal changes and inflammation, thereby playing a key role in the mechanisms of disease progression.

Abstract 2175: Vietnamese medicinal plant compounds show potent anti-pancreatic cancer activityin vitro

BACKGROUND: Pancreatic cancer is a devastating disease with a dismal survival rate of less than 7%. This is due to its late diagnosis and aggressive, metastatic disease. Currently treatment typically consists of surgery and/or treatment with gemcitabine; gemcitabine/nab-paclitaxel or FOLFIRINOX - all chemotherapeutic agents that have natural product-derived components that only prolong survival by 5-6 months at best. As traditional Vietnamese medicinal plants have been used to treat a number of diseases for several thousands of years, there is great potential to investigate their importance as sources of novel therapeutic agents for pancreatic cancer. METHODS: Pancreatic cancer cell lines (BxPC3, MiaPaCa2 and CFPAC1) and the normal pancreatic ductal epithelial (HPDE) cell line were treated with pure compounds isolated from the Vietnamese plants Croton tonkinensis and Salacia cochinchinensis and the effects on cell viability (CCK-8 assay) and apoptosis (caspase-3/-7 activation assay) were assessed to determine their anti-cancer capacity. RESULTS: Treatment of pancreatic cancer cell lines with Pristimerin (from Salacia cochinchinensis) and CT1 (from Croton tonkinensis) resulted in significant dose-dependent growth inhibition. Pancreatic cancer cell lines showed significantly greater sensitivity to Pristimerin treatment after 24 and 72hrs, with a 1.7 – 2.3 fold lower IC50 value in MiaPaCa2 pancreatic cancer cells compared to normal HPDE cells, respectively (24hrs: 48 vs. 111nM; 72hrs: 63.85 vs. 111nM). Significant cytotoxicity was also seen in other pancreatic cancer cell lines (BxPC3 103 - 210nM; CFPAC1 77nM). Treatment with CT1 also resulted in significant dose-dependent cytotoxicity with an IC50 value that was 1.5 – 1.8 fold lower in MiaPaCa2 cells compared to normal HPDE cells, respectively (24hrs: 116 vs. 177nM; 72hrs: 46.5 vs. 84.5nM). Treatment of BxPC3 and CFPAC1 cells with CT1 also led to significant cytotoxicity with IC50 values of 300nM and 170nM, respectively. Moreover, treatment with Pristimerin and CT1 at concentrations of 625nM and 1μM induced caspase-dependent apoptosis of MiaPaCa2 and HPDE cells after 22hrs of treatment. CONCLUSIONS: Pure compounds from Vietnamese medicinal plants show significant pancreatic cancer growth inhibition and induce apoptosis in vitro at concentrations that have minimal effect on normal pancreatic cells and therefore show promise as novel anti-pancreatic cancer therapies. Note: This abstract was not presented at the meeting. Citation Format: Danielle Bond, Phuong Thien Thuong, Do Thi Ha, Nguyen Minh Khoi, Judith Weidenhofer, Christopher J. Scarlett. Vietnamese medicinal plant compounds show potent anti-pancreatic cancer activity in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2175. doi:10.1158/1538-7445.AM2017-2175

The olive phenolic compounds apigenin, luteolin and oleuropein induce cell-cycle arrest and apoptosis in pancreatic cancer cells in vitro

A. BAJRAKTAREVIC, B. KRDZALIC, R. MERDZANIC, L. KUMASIN, A. SELIMOVIC, D. ABDUZAIMOVIC, A. ABDUZAIMOVIC, D. GRANOV ROKOLJ, I. SULJEVIC, H. CUBRO, F. KRUPIC Public Health Institution of Health Center Sarajevo, Pediatrics Department, Sarajevo, Bosnia and Herzegovina, Pediatrics Clinik Jezero, Pulmonology Departement, Sarajevo, Bosnia and Herzegovina, Private laboratory Jelah, Biochemistery Department, Tesanj, Bosnia and Herzegovina, Clinical Medical Center Sarajevo, Biochemistry and Microbiology Laboratory, Sarajevo, Bosnia and Herzegovina, and Institute of Clinical SciencesSahlgrenska AcademyUniversity of Gothenburg, Department of Orthopaedics, Gothenburg, Sweden

Abstract 4364: Posttranscriptional regulation of tetraspanins CD151 & CD9 in breast & prostate cancers

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Rationale The tetraspanins CD151 & CD9 are known to play a prominent role in cancer progression by modulating cell adhesion and migration/invasion. CD151 is commonly overexpressed (metastasis enhancer) and tends to predict poor prognosis in many solid malignancies, whereas the metastasis suppressor CD9 is typically down-regulated which correlates with poor patient outcomes. However, progress in developing CD151 and CD9 as prognostic biomarkers & therapeutic targets has been hampered by a lack of understanding of how they are regulated in normal cells and how they are deregulated in cancers. Objective Many micro-RNAs are deregulated in prostate and breast cancers. Bio-informatic analysis has identified 250 miRNAs that potentially regulate CD151 or CD9. Therefore, we investigated whether miRNAs regulate CD151 and CD9 in a range of normal and cancerous breast and prostate cell lines in vitro. Methods & Results A dual luciferase reporter assay using the 3′UTR of CD151 and CD9 provided evidence that breast & prostate cancer cell lines endogenously express miRNA that regulate tetraspanins and that the extent of regulation varies across the cell lines. Analysis of miRNA expression in normal and cancer cell lines using miRNA arrays and qPCR has confirmed that some miRNAs which are predicted to target CD9, are up-regulated in many prostate and/or breast cancer cell lines compared to non-tumourigenic cells. Furthermore, miRNAs were identified with altered expression that were predicted to target CD151. These miRNA have also been shown to directly regulate CD151 or CD9 using the dual luciferase 3′UTR reporter assay with miRNA mimics. Conclusion Altered miRNA expression may contribute to prostate and breast cancers by manipulating tetraspanins to allow cancer progression and metastasis. Therefore, miRNA may be effective prognostic and/or therapeutic targets for prostate and breast cancers in the future. Citation Format: Danielle R. Bond, Crystal Passfield, Murray Cairns, Leonie K. Ashman, Judith Weidenhofer. Posttranscriptional regulation of tetraspanins CD151 & CD9 in breast & prostate cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4364. doi:10.1158/1538-7445.AM2014-4364