Jennifer A. Higgins

Curcumin inhibits cancer stem cell phenotypes in ex vivo models of colorectal liver metastases, and is clinically safe and tolerable in combination with FOLFOX chemotherapy

Highlights • Curcumin + FOLFOX inhibits growth of primary cancer stem cell (CSC) spheroid models.• Curcumin + FOLFOX decreases expression of CSC markers in primary CSC spheroid models.• Curcumin enhances proapoptotic effects of chemotherapy in explant culture.• Curcumin is safe and tolerable in combination with FOLFOX chemotherapy.• Curcumin is perceived by patients as an acceptable daily adjunct to chemotherapy.

Inter-laboratory variation in DNA damage using a standard comet assay protocol

There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol, which were not related to the level of experience. Therefore, the inter-laboratory variation in DNA damage was only analysed using the results from laboratories that had obtained complete data with the standard comet assay protocol. This analysis showed that the differences between reported levels of DNA SBs/alkaline labile sites in MNBCs were not reduced by applying the standard assay protocol as compared with the laboratorys own protocol. There was large inter-laboratory variation in FPG-sensitive sites by the laboratory-specific protocol and the variation was reduced when the samples were analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained poor results using the standard procedure. This study indicates that future comet assay validation trials should take steps to evaluate the implementation of standard procedures in participating laboratories.

An ECVAG inter-laboratory validation study of the comet assay: inter-laboratory and intra-laboratory variations of DNA strand breaks and FPG-sensitive sites in human mononuclear cells

The alkaline comet assay is an established, sensitive method extensively used in biomonitoring studies. This method can be modified to measure a range of different types of DNA damage. However, considerable differences in the protocols used by different research groups affect the inter-laboratory comparisons of results. The aim of this study was to assess the inter-laboratory, intra-laboratory, sample and residual (unexplained) variations in DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites measured by the comet assay by using a balanced Latin square design. Fourteen participating laboratories used their own comet assay protocols to measure the level of DNA strand breaks and FPG-sensitive sites in coded samples containing peripheral blood mononuclear cells (PBMC) and the level of DNA strand breaks in coded calibration curve samples (cells exposed to different doses of ionising radiation) on three different days of analysis. Eleven laboratories found dose-response relationships in the coded calibration curve samples on two or three days of analysis, whereas three laboratories had technical problems in their assay. In the coded calibration curve samples, the dose of ionising radiation, inter-laboratory variation, intra-laboratory variation and residual variation contributed to 60.9, 19.4, 0.1 and 19.5%, respectively, of the total variation. In the coded PBMC samples, the inter-laboratory variation explained the largest fraction of the overall variation of DNA strand breaks (79.2%) and the residual variation (19.9%) was much larger than the intra-laboratory (0.3%) and inter-subject (0.5%) variation. The same partitioning of the overall variation of FPG-sensitive sites in the PBMC samples indicated that the inter-laboratory variation was the strongest contributor (56.7%), whereas the residual (42.9%), intra-laboratory (0.2%) and inter-subject (0.3%) variations again contributed less to the overall variation. The results suggest that the variation in DNA damage, measured by comet assay, in PBMC from healthy subjects is assay variation rather than variation between subjects.

DNA damage of macrophages at an air-tissue interface induced by metal nanoparticles

Abstract An air-tissue interface model was used to assess nanoparticulate-induced DNA damage to airway macrophages. Human Mono Mac 6 cells and rat alveolar macrophages were cultured on a collagen membrane and the deposition of metal nanoparticles in nitrogen enhanced using electrostatic charge. Cells were exposed to nanoparticles of iron, gold, silver for up to 10 min, then cultured in medium for 24 hours. Damage to DNA was assessed using the Comet assay. Nanoparticle dose delivered to cells varied with metal. Significant DNA damage to macrophages was induced by all three metal nanoparticles. Transmission electron microscopy showed deposition of discrete nanoparticles of gold and silver, but not iron. We conclude that an air-tissue model is a useful method for modelling DNA damage to airway cells from inhalation of metal nanoparticles.

Anthocyans as tertiary chemopreventive agents in bladder cancer: anti-oxidant mechanisms and interaction with mitomycin C

Bladder cancer is associated with high rates of recurrence making tertiary chemoprevention an attractive intervention strategy. Anthocyanins have been shown to possess chemopreventive properties and are detectable in urine after oral ingestion, with higher concentrations achievable via intravesical administration alongside current chemotherapeutic regimens. Yet their apparent ability to protect against certain DNA damage may in turn interfere with cancer treatments. Our aim was therefore to determine the potential of anthocyanins as chemopreventive agents in bladder cancer, their mode of action and effects, both alone and in combination with mitomycin C (MMC). In this study we showed that mirtoselect, a standardised mixture of anthocyanins, possesses significant anti-proliferative activity, causing growth inhibition and apoptosis in bladder cancer cell lines. The anti-oxidative potential of mirtoselect was examined and revealed significantly fewer H2O2-induced DNA strand breaks, as well as oxidised DNA bases in pre-treated cells. In contrast, endogenous levels of oxidised DNA bases were unaltered. Investigations into the possible protective mechanisms associated with these anti-oxidant properties revealed that mirtoselect chelates metal ions. In mirtoselect/MMC combination studies, no adverse effects on measures of DNA damage were observed compared to treatment with MMC alone and there was evidence of enhanced cell death. Consistent with this, significantly more DNA crosslinks were formed in cells treated with the combination. These results show that mirtoselect exerts effects consistent with chemopreventive properties in bladder cancer cell lines and most importantly does so without adversely affecting the effects of drugs used in current treatment regimens. We also provide evidence that mirtoselects anti-oxidative mechanism of action is via metal ion chelation. Overall these results suggest that mirtoselect could be an effective chemopreventive agent in bladder cancer and provides the necessary pre-clinical data for future in vivo animal studies and clinical trials.

Characterization and Propagation of Tumor Initiating Cells Derived from Colorectal Liver Metastases: Trials, Tribulations and a Cautionary Note

Tumor initiating cells (TIC) are increasingly being put forward as a potential target for intervention within colorectal cancer. Whilst characterisation and outgrowth of these cells has been extensively undertaken in primary colorectal cancers, few data are available describing characteristics within the metastatic setting. Tissue was obtained from patients undergoing surgical resection for colorectal liver metastases, and processed into single cell suspension for assessment. Tumor initiating cells from liver metastases were characterised using combinations of EPCAM, Aldehyde dehydrogenase activity, CD133 and CD26. CD133 expression was significantly lower in patients who had received chemotherapy, but this was accounted for by a decrease observed in the male patient cohort only. ALDHhigh populations were rare (0.4 and 0.3% for EPCAM+/ALDHhigh/CD133- and EPCAM+/ALDHhigh/CD133+ populations respectively) and below the limits of detection in 28% of samples. Spheroid outgrowth of metastatic tumor cells across all samples could not be readily achieved using standard spheroid-formation techniques, thus requiring further method validation to reliably propagate cells from the majority of tissues. Spheroid formation was not enhanced using additional growth factors or fibroblast co-culture, but once cells were passaged through NOD-SCID mice, spheroid formation was observed in 82% samples, accompanied by a significant increase in CD26. Order of spheroid forming ability was ALDHhigh>CD133>CD26. Samples sorted by these markers each had the ability to reform ALDHhigh, CD133 and CD26 positive populations to a similar extent, suggestive of a high degree of plasticity for each population. Ex vivo TIC models are increasingly being utilised to assess efficacy of therapeutic interventions. It is therefore essential that such investigations use well-characterised models that are able to sustain TIC populations across a large patient cohort in order that the inherent heterogeneity observed in cancer populations is maintained.

Abstract 258: Targeting Nanog in 3D explant models for the evaluation of cancer prevention agents

Background: The transcription factor Nanog is crucial for the self-renewal of cancer stem-like cells (CSCs). Nanog expression in colorectal cancer (CRC) tissue correlates with lymph node metastasis and poor prognosis. Since Nanog is not expressed in most tissues, including normal adult stem-cells, it represents a therapeutic target specific to cancer cells. Curcumin inhibits proliferation and expansion of CSCs derived from CRC and adenomas. In NOD/SCID mice bearing xenografts from patient-derived CRC CSCs, curcumin significantly inhibited tumour growth and improved survival. In addition, curcumin binds directly to Nanog recombinant protein (quantified using microscale thermophoresis). We have developed 3D in-vitro primary human explant models to further characterise the effects of curcumin on Nanog. In this work the hypothesis is tested that Nanog may be an early marker of response for CRC prevention agents. Methods: Patient-derived CRC and adenoma tissue was cubed (2x2x2mm) and treated for 24 hours with curcumin. Following treatment, explant tissues were processed for analysis by immunohistochemistry (IHC) (n=6) and flow cytometry (n=17). The effect of curcumin on CSCs (defined by expression of aldehyde dehydrogenase (ALDH) or Nanog) and differentiation (via Mucin 2 expression) was analysed using IHC. Additionally, cells expressing Nanog (Nanog+) or Nanog plus proliferation marker Ki67 (Nanog+Ki67+) were assessed using flow cytometry. Results: A range of adenoma (n=5) and Dukes stage A-C CRC (n=18) samples were studied. Following exposure to curcumin, a 30% reduction was observed in Nanog+ and Nanog+Ki67+ cells. Nanog+ cell number was decreased in a curcumin concentration-dependent fashion in 6 samples and concentration-independently in a further 8. No response was observed in 3 samples. A reduction in Nanog and ALDH with concurrent increase in differentiation was observed via IHC in one sample. Conclusion: Our data suggest Nanog is targeted by curcumin in adenoma and CRC tissues. Nanog may serve as a biomarker in clinical trials to identify individuals most amenable to treatment with curcumin alone or in combination treatment. Crucially, this will help select those who are likely to benefit from curcumin as a cancer prevention agent. Ultimately, this concept may be applicable to the evaluation of novel CRC prevention agents. Citation Format: Sam Khan, Ankur Karmokar, Zahirah Sidat, Nalini Foreman, David Moore, Jennifer Higgins, Emma Parrott, Despoina Theofanous, Dominic Hobbs, Lynne Howells, Anne Thomas, Karen Brown. Targeting Nanog in 3D explant models for the evaluation of cancer prevention agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 258.