Sarah Haywood-Small

Aldehyde dehydrogenase activity selects for the holoclone phenotype in prostate cancer cells

Aldehyde dehydrogenase 1 (ALDH) activity is considered to be a marker of cancer stem cells (CSCs) in many tumour models, since these cells are more proliferative and tumourigenic than ALDH(Lo) cells in experimental models. However it is unclear whether all CSC-like cells are within the ALDH(Hi) population, or whether all ALDH(Hi) cells are highly proliferative and tumourigenic. The ability to establish a stem cell hierarchy in vitro, whereby sub-populations of cells have differing proliferative and differentiation capacities, is an alternate indication of the presence of stem cell-like populations within cell lines. In this study, we have examined the interaction between ALDH status and the ability to establish a stem cell hierarchy in PC3 prostate cancer cells. We demonstrate that PC3 cells contain a stem cell hierarchy, and isolation of ALDH(Hi) cells enriches for the most primitive holoclone population, however holoclone formation is not restricted to ALDH(Hi) cells. In addition, we show that ALDH activity undergoes phenotypic plasticity, since the ALDH(Lo) population can develop ALDH(Hi) populations comparable to parental cells within 2 weeks in culture. Furthermore, we show that the majority of ALDH(Hi) cells are found within the least primitive paraclone population, which is circumvented by culturing PC3 cells as spheroids in defined medium favouring stem cell characteristics. Although ALDH(Hi) status enriches for holoclone formation, this activity may be mediated by a minority of ALDH(Hi) cells.

Differential Effects of Polyphenols on Proliferation and Apoptosis in Human Myeloid and Lymphoid Leukemia Cell Lines

Background: Mortality rates for leukemia are high despite considerable improvements in treatment. Since polyphenols exert pro-apoptotic effects in solid tumors, our study investigated the effects of polyphenols in haematological malignancies. The effect of eight polyphenols (quercetin, chrysin, apigenin, emodin, aloe-emodin, rhein, cis-stilbene and trans-stilbene) were studied on cell proliferation, cell cycle and apoptosis in four lymphoid and four myeloid leukemic cells lines, together with normal haematopoietic control cells. Methods: Cellular proliferation was measured by CellTiter-Glo® luminescent assay; and cell cycle arrest was assessed using flow cytometry of propidium iodide stained cells. Apoptosis was investigated by caspase-3 activity assay using flow cytometry and apoptotic morphology was confirmed by Hoescht 33342 staining. Results: Emodin, quercetin, and cis-stilbene were the most effective polyphenols at decreasing cell viability (IC50 values of 5-22 µM, 8-33 µM, and 25-85 µM respectively) and inducing apoptosis (AP50 values (the concentration which 50% of cells undergo apoptosis) of 2-27 µM, 19-50 µM, and 8-50 µM respectively). Generally, lymphoid cell lines were more sensitive to polyphenol treatment compared to myeloid cell lines, however the most resistant myeloid (KG-1a and K562) cell lines were still found to respond to emodin and quercetin treatment at low micromolar levels. Non-tumor cells were less sensitive to all polyphenols compared to the leukemia cells. Conclusions: These findings suggest that polyphenols have anti-tumor activity against leukemia cells with differential effects. Importantly, the differential sensitivity of emodin, quercetin, and cis-stilbene between leukemia and normal cells suggests that polyphenols are potential therapeutic agents for leukemia.

Recombinant "IMS TAG" proteins - A new method for validating bottom-up matrix-assisted laser desorption/ionisation ion mobility separation mass spectrometry imaging

RATIONALE Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) provides a methodology to map the distribution of peptides generated by in situ tryptic digestion of biological tissue. It is challenging to correlate these peptides to the proteins from which they arise because of the many potentially overlapping and hence interfering peptide signals generated. METHODS A recombinant protein has been synthesised that when cleaved with trypsin yields a range of peptide standards for use as identification and quantification markers for multiple proteins in one MALDI-IMS-MSI experiment. Mass spectrometry images of the distribution of proteins in fresh frozen and formalin-fixed paraffin-embedded tissue samples following in situ tryptic digestion were generated by isolating signals on the basis of their m/z value and ion mobility drift time, which were correlated to matching peptides in the recombinant standard. RESULTS Tryptic digestion of the IMS-TAG protein and MALDI-MS analysis yielded m/z values and ion mobility drift time for the signature peptides included in it. MALDI-IMS-MSI images for the distribution of the proteins HSP90 and vimentin, in FFPE EMT6 mouse tumours, and HSP90 and plectin in a fresh frozen mouse fibrosarcoma, were generated by extracting ion images at the corresponding m/z value and drift time from the tissue samples. CONCLUSIONS The IMS-TAG approach provides a new means to confirm the identity of peptides generated by in situ digestion of biological tissue.

Polyphenols act synergistically with doxorubicin and etoposide in leukaemia cell lines

The study aimed to assess the effects of polyphenols when used in combination with doxorubicin and etoposide, and to determine whether polyphenols sensitised leukaemia cells, causing inhibition of cell proliferation, cell cycle arrest and induction of apoptosis. This study is based on findings in solid cancer tumours, which have shown that polyphenols can sensitize cells to chemotherapy, and induce apoptosis and/or cell-cycle arrest. This could enable a reduction of chemotherapy dose and off-target effects, whilst maintaining treatment efficacy. Quercetin, apigenin, emodin, rhein and cis-stilbene were investigated alone and in combination with etoposide and doxorubicin in two lymphoid and two myeloid leukaemia cells lines. Measurements were made of ATP levels (using CellTiter-Glo assay) as an indication of total cell number, cell cycle progression (using propidium iodide staining and flow cytometry) and apoptosis (NucView caspase 3 assay and Hoechst 33342/propidium iodide staining). Effects of combination treatments on caspases 3, 8 and 9 activity were determined using Glo luminescent assays, glutathione levels were measured using the GSH-Glo Glutathione Assay and DNA damage determined by anti-γH2AX staining. Doxorubicin and etoposide in combination with polyphenols synergistically reduced ATP levels, induced apoptosis and increased S and/or G2/M phase cell cycle arrest in lymphoid leukaemia cell lines. However, in the myeloid cell lines the effects of the combination treatments varied; doxorubicin had a synergistic or additive effect when combined with quercetin, apigenin, emodin, and cis-stilbene, but had an antagonistic effect when combined with rhein. Combination treatment caused a synergistic downregulation of glutathione levels and increased DNA damage, driving apoptosis via caspase 8 and 9 activation. However, in myeloid cells where antagonistic effects were observed, this was associated with increased glutathione levels and a reduction in DNA damage and apoptosis. This study has demonstrated that doxorubicin and etoposide activity were enhanced by polyphenols in lymphoid leukaemia cells, however, differential responses were seen in myeloid cells with antagonistic responses seen in some combination therapies.

A qualitative exploration of the experiences of living with and being treated for fibromyalgia

This study explores the life and treatment experience of people in the United Kingdom with fibromyalgia in order to inform the development of treatments which are both effective and acceptable to users. Qualitative interviews were conducted with 14 participants with interpretative phenomenological analysis used as the theoretical framework and analytical method. The themes identified were as follows: Inauthenticity of fibromyalgia, An Unconventional healthcare experience, Re-creating support networks, Challenging the working identity, Threatening the family dynamic and Fighting, accepting or accommodating? The biopsychosocial impacts of fibromyalgia disrupted the identity, lifestyle, roles and relationships of our participants with such challenges further exacerbated by the contested nature of the illness.

Molecular characterisation of the monocytic cell line THP-1 demonstrates a discrepancy with the documented HLA type.

Dear Editor, The use of the acute monocytic leukemia cell line, THP-1, has been well documented since its establishment was reported more than 30 years ago in the IJC. Since then, the THP-1 cell line has been used in countless research papers and is now a widely recognized resource as a model of monocyte cells within the immunology and oncology research communities. Routinely applied good cell culture practice of THP-1, as well as all other cell lines, requires quality assurance of the cell line, including assessment of relevant phenotypes and genotypes, state of differentiation and the assessment of any possible crosscontamination. In line with these requirements, our recent use of the THP-1 cell line revealed an inconsistency with the reported Human Leukocyte Antigen (HLA) type which may have potential consequences for some researchers. Initially THP-1 was recovered from our bank of frozen cell lines and cultured in RPMI 1640 medium (5% Fetal Calf Serum (FCS), 200 mM L-Glut, 100 lg/ml Pen/Strep) before HLA typing for HLA class I A and B, and the HLA class II DR and DQ antigens, via commercially available serological typing trays. These serological typing trays represent the contemporary equivalent of the assay used to determine the HLA type of THP-1 when the cell line was established. The results of these assays conclusively identified HLA-A2. ‘‘Weak’’ reactions against other class I antigens and crossreactivity, particularly between HLA-B5 and B15 antisera, confounded the identification of a full HLA-A and B type. The HLA class II antigens were entirely inconclusive. Since the THP-1 cell line was established in 1980, there have been marked advances within the detection methodologies used to determine HLA types, most notably the development of molecular biology-based techniques, such as the polymerase chain reaction (PCR). Therefore, to determine a conclusive HLA type for the THP-1 cell line, an ‘‘in-house’’ PCR using sequence specific primers (PCR-SSP) was performed for HLA-A*, B*, C*, DRB1*, DRB3*, DRB4*, DRB5* and DQB1* genes. In addition, an ABO group was determined using a similar method. Both of these in-house genotyping systems are based on methods which have been previously published. Using this assay, we identified the following HLA type for THP-1. HLA-A*02; B*15; C*03; DRB1*01, DRB1*15; DRB5*01/02; DQB1*05 and DQB1*06 (and ABO blood group genotype BB). The reported HLA type for THP-1 is HLA-A2, A9, B5, DRw1 and DRw2. (The HLA nomenclature system used here corresponds to the contemporary system at the time THP-1 was established in 1980). Clearly the HLA class I type did not correspond to the reported HLA type for THP-1, differing at the HLA class I A and B loci (to our knowledge HLA-C* has not previously been reported for THP-1). The HLA class II type corresponded to the original HLA type of HLA-DRw1 and DRw2, being DRB1*01 and DRB1*15. The expressed product of the HLA-DRB1*15 allele represents a split of the DR2 antigen and demonstrates the increased resolving capabilities of the PCR-SSP assay in comparison to the serological technique. (again to our knowledge HLA-DQB1* has not previously been reported). In contrast to the reported type, THP-1 appeared to be homozygous at the HLA-A, B and C loci by PCR-SSP. Alterations in the HLA class I phenotype of malignant cells is a frequent event during cancer progression, allowing tumor cells to evade the immune system. Indeed, loss of one major histocompatibility complex class I haplotype in human melanoma cells has been shown not only to allow evasion of immunosurveillance but also to increase their intrinsic oncogenic potential. The mechanisms which lead to HLA class I alterations can occur at any step required for HLA synthesis. Most commonly, the alteration represents a structural defect or a regulatory defect on the transcriptional level. Such alterations would not interfere with the PCR-SSP method used here, although a rarer deletion event upon chromosome 6 would be undetectable. In addition the ‘‘missing’’ class I HLA antigens from the original report show strong crossreactivity with the confirmed HLA type, and most likely account for this discrepancy, indeed our serological assay also demonstrated crossreactivity at the HLA-B5 and B15 antigens, which were proved erroneous by the PCR-SSP. Interestingly, the HLA-B*15 identified within the haplotype HLA-A*02; B*15; C*03, is unusual in itself, corresponding to the serological equivalent of B75 (15). The PCR-SSP was capable of resolving the B*15 allele to B*15:08 or B*15:11. A search on the Allele* Frequencies in World Populations website for each of these alleles revealed their frequency within specific populations. Of the two alleles B*15:11 appeared to be the more common, as, although extremely rare within the majority of populations, it is seen in the Le tt er to th e E di to r

SAHA-induced TRAIL-sensitisation of Multiple Myeloma cells is enhanced in 3D cell culture

Background Multiple Myeloma (MM) is currently incurable despite many novel therapies. Tumour Necrosis Factor‐Related Apoptosis‐Inducing Ligand (TRAIL) is a potential anti‐tumour agent although effects as a single agent are limited. In this study, we investigated whether the Histone Deacetylase (HDAC) inhibitor SAHA can enhance TRAIL‐induced apoptosis and target TRAIL resistance in both suspension culture, and 3D cell culture as a model of disseminated MM lesions that form in bone. Methods The effects of SAHA and/or TRAIL in 6 Multiple Myeloma cell lines were assessed in both suspension cultures and in an Alginate‐based 3D cell culture model. The effect of SAHA and/or TRAIL was assessed on apoptosis by assessment of nuclear morphology using Hoechst 33342/Propidium Iodide staining. Viable cell number was assessed by CellTiter‐Glo luminescence assay, Caspase‐8 and ‐9 activities were measured by Caspase‐Glo™ assay kit. TRAIL‐resistant cells were generated by culture of RPMI 8226 and NCI‐H929 by acute exposure to TRAIL followed by selection of TRAIL‐resistant cells. Results TRAIL significantly induced apoptosis in a dose‐dependent manner in OPM‐2, RPMI 8226, NCI‐H929, U266, JJN‐3 MM cell lines and ADC‐1 plasma cell leukaemia cells. SAHA amplified TRAIL responses in all lines except OPM‐2, and enhanced TRAIL responses were both via Caspase‐8 and ‐9. SAHA treatment induced growth inhibition that further increased in the combination treatment with TRAIL in MM cells. The co‐treatment of TRAIL and SAHA reduced viable cell numbers all cell lines. TRAIL responses were further potentiated by SAHA in 3D cell culture in NCI‐H929, RPMI 8226 and U266 at lower TRAIL + SAHA doses than in suspension culture. However TRAIL responses in cells that had been selected for TRAIL resistance were not further enhanced by SAHA treatment. Conclusions SAHA is a potent sensitizer of TRAIL responses in both TRAIL sensitive and resistant cell lines, in both suspension and 3D culture, however SAHA did not sensitise TRAIL‐sensitive cell populations that had been selected for TRAIL‐resistance from initially TRAIL‐sensitive populations. SAHA may increase TRAIL sensitivity in insensitive cells, but not in cells that have specifically been selected for acquired TRAIL‐resistance. HighlightsThe HDAC inhibitor SAHA sensitised Myeloma cells to TRAIL‐induced apoptosis.TRAIL, SAHA and combination apoptotic responses are potentiated in 3D cell culture.TRAIL‐resistant cells are not further sensitised to TRAIL‐induced apoptosis by SAHA.

Glutathione is key to the synergistic enhancement of doxorubicin and etoposide by polyphenols in leukaemia cell lines

Recently published in Nature: Cell Death and Discovery, Mahbub et al.1 have demonstrated that polyphenols can synergistically enhance the action of the topoisomerase II inhibitors: doxorubicin and etoposide in leukaemia cells. A reduction of glutathione (GSH) was strongly associated with sensitising cells to the pro-apoptotic effects of polyphenols when used in combination with doxorubicin or etoposide. Importantly, when polyphenols and topoisomerase II inhibitors were combined, it was possible to induce a synergistic decrease in cell proliferation (measured as ATP levels), cell-cycle arrest and induction of apoptosis in leukaemia cell lines.

Patients’ perspective of the effectiveness and acceptability of pharmacological and non-pharmacological treatments of fibromyalgia

Abstract Background and aims Fibromyalgia is a complex condition characterised by widespread pain, sleep disturbance, fatigue and cognitive impairment, with a global mean prevalence estimated at 2.7%. There are inconsistencies in guidelines on the treatment of fibromyalgia leading to dissatisfaction from patients and healthcare professionals. This study investigated patient-reported outcomes of pharmacological and non-pharmacological treatment usage and effectiveness with an assessment of acceptability. Methods Nine hundred and forty-one participants completed a self-administered anonymous questionnaire giving quantitative data of demographics, treatment usage and treatment outcomes. Participant-reported effectiveness and side effects were compared in the following treatment classes: analgesics, antidepressants, gabapentinoids, gastrointestinal treatments, activity interventions, dietary-based treatments, and psychological, physical and alternative therapies. Participants also reported whether they knew about or had tried different treatments. Results The results from the online survey indicated that the range of mean effectiveness ratings were similar for pharmacological and non-pharmacological treatments, whereas non-pharmacological treatments had lower side effects ratings and higher acceptability relative to pharmacological treatments. Participants were not aware of some treatment options. Conclusions The results show lower side effects ratings and higher acceptability for non-pharmacological treatments compared to pharmacological treatments despite similar effectiveness ratings. Implications This article presents results from a large online survey on fibromyalgia patient perspectives of pharmacological and non-pharmacological treatments. Results will inform healthcare professionals and patients about optimal treatments based on ratings of effectiveness, side effects and acceptability that are tailored to patient symptom profiles. Some participants were unaware of treatment options highlighting the importance of patient education allowing collaboration between patients and healthcare professionals to find optimal treatments.

What causes Fibromyalgia? An online survey of patient perspectives.

Fibromyalgia is a severe chronic pain condition that affects every aspect of life. Causes of the condition remain unclear, and quantitative research cannot account for patients’ personal illness narratives and perceptions. This online survey gathered qualitative accounts of the perceived causes of their condition from 596 people with fibromyalgia, which were analyzed thematically. Themes were “Bodily assault, ill-health, and change”; “Emotional trauma and distress”; “Stress and vulnerability”; and “Explaining and authenticating fibromyalgia.” Discussion focuses on the complexity of causation, the importance of understanding and having symptoms validated, and the potential for benefiting from patient expertise in building better practitioner–client relationships.