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Abstract Background Mononuclear platinum anticancer agents hold a pivotal place in the treatment of many forms of cancers, however, there is a potential to improve response to evade resistance development and toxic side effects. BBR3464 is a promising trinuclear platinum anticancer agent, which is a polyamine mimic. The aim was to investigate the influence of polyamine pool reduction on the cytotoxic effects of the trinuclear platinum complex BBR3464 and cisplatin. Polyamine pool reduction was achieved by treating cells with either the polyamine biosynthesis inhibitor α-difluoromethylornithine (DFMO) or the polyamine analogue N1,N11-diethylnorspermine (DENSPM). Methods A human squamous cell carcinoma cell line, LU-HNSCC-4, established from a primary head and neck tumour was used to evaluate cellular effects of each drug alone or combinations thereof. High-performance liquid-chromatography was used to quantify intracellular polyamine contents. Inductively coupled mass spectroscopy was used to quantify intracellular platinum uptake. Cells were exposed to DFMO or DENSPM during 48 h at concentrations ranging from 0 to 5 mM or 0 to 10 μM, respectively. Thereafter, non-treated and treated cells were exposed to cisplatin or BBR3464 during 1 h at concentrations ranging from 0 to 100 μM. A 96-well assay was used to determine cytotoxicity after five days after treatment. Results The cytotoxic effect of BBR3464 on LU-HNSCC-4 cells was increased after cells were pre-treated with DENSPM or DFMO, and the interaction was found to be synergistic. In contrast, the interaction between cisplatin and DFMO or DENSPM was near-additive to antagonistic. The intracellular levels of the polyamines putrescine and spermidine were decreased after treatment with DFMO, and treatment with DENSPM resulted in an increase in putrescine level and concomitant decrease in spermidine and spermine levels. The uptake of BBR3464 was significantly increased after pre-treatment of the cells with DFMO, and varied dependent on the concentration of DENSPM. The uptake of cisplatin was unchanged. Conclusions Taken together, these results demonstrate that combinations of polyamine synthesis inhibitors with BBR3464 appear to be a promising approach to enhance the anticancer activity against HSCC.

Abstract Background Overcrowding in emergency departments is a worldwide problem. A systematic literature review was undertaken to scientifically explore which interventions improve patient flow in emergency departments. Methods A systematic literature search for flow processes in emergency departments was followed by assessment of relevance and methodological quality of each individual study fulfilling the inclusion criteria. Studies were excluded if they did not present data on waiting time, length of stay, patients leaving the emergency department without being seen or other flow parameters based on a nonselected material of patients. Only studies with a control group, either in a randomized controlled trial or in an observational study with historical controls, were included. For each intervention, the level of scientific evidence was rated according to the GRADE system, launched by a WHO-supported working group. Results The interventions were grouped into streaming, fast track, team triage, point-of-care testing (performing laboratory analysis in the emergency department), and nurse-requested x-ray. Thirty-three studies, including over 800,000 patients in total, were included. Scientific evidence on the effect of fast track on waiting time, length of stay, and left without being seen was moderately strong. The effect of team triage on left without being seen was relatively strong, but the evidence for all other interventions was limited or insufficient. Conclusions Introducing fast track for patients with less severe symptoms results in shorter waiting time, shorter length of stay, and fewer patients leaving without being seen. Team triage, with a physician in the team, will probably result in shorter waiting time and shorter length of stay and most likely in fewer patients leaving without being seen. There is only limited scientific evidence that streaming of patients into different tracks, performing laboratory analysis in the emergency department or having nurses to request certain x-rays results in shorter waiting time and length of stay.

Abstract Emergency department (ED) triage is used to identify patients' level of urgency and treat them based on their triage level. The global advancement of triage scales in the past two decades has generated considerable research on the validity and reliability of these scales. This systematic review aims to investigate the scientific evidence for published ED triage scales. The following questions are addressed: 1. Does assessment of individual vital signs or chief complaints affect mortality during the hospital stay or within 30 days after arrival at the ED? 2. What is the level of agreement between clinicians' triage decisions compared to each other or to a gold standard for each scale (reliability)? 3. How valid is each triage scale in predicting hospitalization and hospital mortality? A systematic search of the international literature published from 1966 through March 31, 2009 explored the British Nursing Index, Business Source Premier, CINAHL, Cochrane Library, EMBASE, and PubMed. Inclusion was limited to controlled studies of adult patients (≥15 years) visiting EDs for somatic reasons. Outcome variables were death in ED or hospital and need for hospitalization (validity). Methodological quality and clinical relevance of each study were rated as high, medium, or low. The results from the studies that met the inclusion criteria and quality standards were synthesized applying the internationally developed GRADE system. Each conclusion was then assessed as having strong, moderately strong, limited, or insufficient scientific evidence. If studies were not available, this was also noted. We found ED triage scales to be supported, at best, by limited and often insufficient evidence. The ability of the individual vital signs included in the different scales to predict outcome is seldom, if at all, studied in the ED setting. The scientific evidence to assess interrater agreement (reliability) was limited for one triage scale and insufficient or lacking for all other scales. Two of the scales yielded limited scientific evidence, and one scale yielded insufficient evidence, on which to assess the risk of early death or hospitalization in patients assigned to the two lowest triage levels on a 5-level scale (validity).

Food components modify the risk of cancer at a large number of sites but the mechanism of action is unknown. In the present investigation, we studied the effect of the peptide lactoferricin derived from bovine milk lactoferrin on human colon cancer CaCo-2 cells. The cells were either untreated or treated with 2.0, 0.2, or 0.02 μM lactoferricin. Cell cycle kinetics were investigated with a bromodeoxyuridine DNA flow cytometric method. The results show that lactoferricin treatment slightly but significantly prolonged the S phase of the cell cycle. Lactoferricin treatment lowered the level of cyclin E1, a protein involved in the regulation of genes required for G1/S transition and consequently for efficient S phase progression. The slight prolongation of the S phase resulted in a reduction of cell proliferation, which became more apparent after a long treatment time. [ABSTRACT FROM AUTHOR]

Since 1958, cell culture media supplemented with fetal bovine serum is used, despite the well-known concerns about animal welfare, reproducibility, reliability, relevance, and safety. To obliterate these concerns and increase scientific accuracy, we recently published an open access, publicly available paper on a defined medium composition to make it possible for any lab to prepare this medium. The medium supports routine culturing and cell banking as well as investigations of growth curves, dose response testing of compounds of cells in 2D and 3D, and cell migration; all important aspects for research and toxicology. Here we give a detailed description of how to mix the defined universal cell culture medium in 14 simple steps to support any entity that wishes to make it. We also list different normal and cancer cell lines that have been cultured in the defined medium.•Open source composition of animal product free universal cell culture medium•Protocols for mixing solutions of small xeno free molecules for supplementation•Protocols for mixing solutions of human proteins for supplementation., (© 2024 The Author(s).)

Cell culturing methods are increasingly used to reduce and replace the use of live animals in biomedical research and chemical toxicity testing. Although live animals are avoided when using cell culturing methods, they often contain animal-derived components of which one of the most commonly used is foetal bovine serum (FBS). FBS is added to cell culture media among other supplements to support cell attachment/spreading and cell proliferation. The safety, batch-to-batch variation, and ethical problems with FBS are acknowledged and therefore world-wide efforts are ongoing to produce FBS free media. Here, we present the composition of a new defined medium with only human proteins either recombinant or derived from human tissues. This defined medium supports long-term culturing/routine culturing of normal cells and of cancer cells, and can be used for freezing and thawing of cells, i.e . for cell banking. Here, we show for our defined medium, growth curves and dose response curves of cells grown in two and three dimensions, and applications such as cell migration. Cell morphology was studied in real time by phase contrast and phase holographic microscopy time-lapse imaging. The cell lines used are human cancer-associated fibroblasts, keratinocytes, breast cancer JIMT-1 and MDA-MB-231 cells, colon cancer CaCo-2 cells, and pancreatic cancer MiaPaCa-2 cells as well as the mouse L929 cell line. In conclusion, we present the composition of a defined medium without animal-derived products which can be used for routine culturing and in experimental settings for normal cells and for cancer cells, i.e. our defined medium provides a leap towards a universal animal product free cell culture medium., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Cell culture techniques are strongly connected with modern scientific laboratories and production facilities. Thus, choosing the most suitable medium for the cells involved is vital, not only directly to optimise cell viability but also indirectly to maximise the reliability of the experiments performed with the cells. Fetal bovine or calf serum (FBS or FCS, respectively) is the most commonly used cell culture medium supplement, providing various nutritional factors and macromolecules essential for cell growth. Yet, the use of FBS encompasses a number of disadvantages. Scientifically, one of the most severe disadvantages is the lot-to-lot variability of animal sera that hampers reproducibility. Therefore, transitioning from the use of these ill-defined, component-variable, inconsistent, xenogenic, ethically questionable and even potentially infectious media supplements, is key to achieving better data reproducibility and thus better science. To demonstrate that the transition to animal component-free cell culture is possible and achievable, we highlight three different scenarios and provide some case studies of each, namely: i) the adaptation of single cell lines to animal component-free culture conditions by the replacement of FBS and trypsin; ii) the adaptation of multicellular models to FBS-free conditions; and (iii) the replacement of FBS with human platelet lysate (hPL) for the generation of primary stem/stromal cell cultures for clinical purposes. By highlighting these examples, we aim to foster and support the global movement towards more consistent science and provide evidence that it is indeed possible to step out of the currently smouldering scientific reproducibility crisis.

The present article describes a study of the effects of alpha-amylase ( α -amylase) on the human neuroblastoma (NB) cell lines SH-SY5Y, IMR-32, and LA-N-1. NB is the most common malignancy diagnosed in infants younger than 12 months. Some clinical observations revealed an inverse association between the risk of NB development and breastfeeding. α -Amylase which is present in breast milk was shown to have anticancer properties already in the beginning of the 20th century. Data presented here show that pancreatic α -amylase inhibits cell proliferation and has a direct impact on glucose uptake in the human NB cell lines. Our results point out the importance of further research which could elucidate the α -amylase mode of action and justify the presence of this enzyme in breast milk as a possible inhibitor of NB development. α -Amylase can be thus recognized as a potential safe and natural mild/host anticancer agent minimizing chemotherapy-related toxicity in the treatment of NB., Competing Interests: Kateryna Pierzynowska is employed by the SGPlus-group, Trelleborg, Sweden. Sofia Thomasson and Stina Oredsson declare no competing interests., (Copyright © 2022 Kateryna Pierzynowska et al.)

Vernonia leopoldi (Sch. Bip. ex Walp.) Vatke (Asteraceae) is one of the widely used anti-cancer traditional medicinal plants in Ethiopia, despite the lack of data to support its therapeutic efficacy. Here we describe the isolation of compounds from the plant and the investigation of their cytotoxicity and other bioactivities. We identified the novel sesquiterpene lactone (SL) 11ß,13-dihydrovernodalol along with the three other SLs (vernomenin, vernolepin, and 11ß,13-dihydrovernodalin) and three flavonoids (apigenin, eriodyctiol, and luteolin) isolated from this plant for the first time. The structures of all the compounds were established based on extensive analysis of nuclear magnetic resonance spectroscopic data and confirmed by high-resolution electrospray ionization mass spectrometry. We then studied the biological activities of the SLs and found that all were cytotoxic at low μM ranges against MCF-7 and JIMT-1 breast cancer cells as well as against the normal-like MCF-10A breast epithelial cells evaluated in a spectrophotometric assay. All the SLs significantly reduced JIMT-1 cell migration after 72 h of treatment with 2 μM concentrations in a wound healing assay. Treatment with all SLs reduced the aldehyde dehydrogenase expressing cancer stem cell sub-population of the JIMT-1 cells significantly, evaluated by flow cytometry. Only 11ß,13-dihydrovernodalin resulted in a significant inhibition of tumor necrosis factor-α-induced translocation of nuclear factor κB to the cell nucleus. In addition, we show that the reporter fluorophore nitrobenzoxadiazole (NBD) can successfully be conjugated with an SL and that this SL-NBD conjugate is taken up efficiently in JIMT-1 cells. Therefore, the overall bioactivities of the SL compounds and specifically their effects against the stemness of breast cancer cells make them prime candidates for further in-depth investigation., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 Published by Elsevier B.V.)

Background: Flavonoids are compounds of interest in the search for new anti-cancer therapies. We have previously isolated the methoxyflavones 5,4'-dihydroxy-6,7,8,3'-tetramethoxyflavone (8-methoxycirsilineol), 5,4'-dihydroxy-6,7,8-trimethoxyflavone (xanthomicrol), and 5,4,'3'-trihydroxy-6,7,8-trimethoxyflavone (sideritoflavone) from Baccharis densiflora. Herein, we investigate the toxicity of these methoxyflavones in human breast-derived cell line. Our main aim was to focus on the cancer stem cell (CSC) sub-population of JIMT-1 breast cancer cells., Methods: Initially, dose response experiments yielding inhibitory concentration 50 (IC 50 ) values were performed using MCF-7, HCC1937, and JIMT-1 breast cancer, and the MCF-10A normal-like breast cell lines to get an understanding of toxic ranges. Due to a clear difference in the toxicity of the flavones, only sideritoflavone was selected for further studies using the JIMT-1 cell line. Effects on the CSC sub-population was investigated using flow cytometry-based methods. A wound healing assay and digital holographic microscopy were used to investigate effects on cell movement. A reporter assay was used to study effects on signal transduction pathways and Western blot for protein expression., Results: The dose response data showed that 8-methoxycirsilineol was non-toxic at concentrations below 100 μM, that the IC 50 of xanthomicrol was between 50 and 100 μM, while sideritoflavone was highly toxic with a single digit μM IC 50 in all cell lines. Treatment of the JIMT-1 cells with 2 μM sideritoflavone did not selectively effect the CSC sub-population. Instead, sideritoflavone treatment inhibited the proliferation of both the non-CSC and the CSC sub-populations to the same extent. The inhibition of cell proliferation resulted in an accumulation of cells in the G 2 phase of the cell cycle and the treated cells showed an increased level of γ-H2A histone family member X indicating DNA double strand breaks. Analysis of the effect of sideritoflavone treatment on signal transduction pathways showed activation of the Wnt, Myc/Max, and transforming growth factor-β pathways. The level of p65/nuclear factor kappa-light-chain-enhancer of activated Β cells was increased in sideritoflavone-treated cells. Cell movement was decreased by sideritoflavone treatment., Conclusions: Altogether our data show that the methoxyflavone sideritoflavone has favourable anti-cancer effects that may be exploited for development to be used in combination with CSC specific compounds.

The appreciation that cell interactions in tissues is dependent on their three dimensional (3D) distribution has stimulated the development of 3D cell culture models. We constructed an artificial 3D tumour by culturing human breast cancer JIMT-1 cells and human dermal fibroblasts (HDFs) in a 3D network of electrospun polycaprolactone fibres. Here, we investigate ECM components produced by the cells in the artificial 3D tumour, which is an important step in validating the model. Immunostaining and confocal fluorescence microscopy show that the ECM proteins fibronectin, collagen I, and laminin are deposited throughout the entire 3D structure. Secreted soluble factors including matrix metalloproteinases (MMPs) and interleukine-6 (IL-6) were analysed in collected medium and were found to be mainly derived from the HDFs. Treatment with transforming growth factor-β1 (TGF-β1), a major cytokine found in a tumour, significantly alters the MMP activity and IL-6 concentration. In addition, TGF-β1 treatment, changes the morphology of the HDFs to become more elongated and with increased linearized actin filaments compared to non-treated HDFs. Collectively, these novel findings suggest that the artificial 3D tumour displays a clear cell distribution and ECM deposition that resembles a tumour environment in vivo, suggesting an innovative biological model to study a human tumour.

Two-dimensional (2D) culturing of cancer cells has been indispensable for the development of anti-cancer drugs. Drug development, however, is lengthy and costly with a high attrition rate, calling to mind that 2D culturing does not mimic the three-dimensional (3D) tumour microenvironment in vivo . Thus, began the development of 3D culture models for cancer research. We have constructed a 3D 96-well plate using electrospun fibres made of biocompatible polycaprolactone (PCL). Finely-cut PCL fibre pieces in water/ethanol solution was pipetted to the wells of hydrophobic 96-well plates. A fibrous network of approximately 200 μm thickness and high porosity was formed after crosslinking and drying. Human JIMT-1 breast cancer cells or fibroblasts were seeded into the network. Confocal microscopy shows that the cells grow throughout the fibre network. The toxicity of paclitaxel and an experimental salinomycin analogue was assessed and compared in 2D and 3D cultures incubated under conditions of normoxia and hypoxia often found in tumours. The toxicity of both compounds is lower when the cells are cultured in 3D compared to 2D in either normoxia or hypoxia. We conclude that our 96-well assay is a cost-efficient tool that may be used for high-throughput pre-clinical screening of potential anti-cancer compounds., Competing Interests: The authors Atena Malakpour-Permlid and Stina Oredsson declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Published by Elsevier B.V.)

Background: The incidence and mortality of breast cancer in women is increasing worldwide. Breast cancer contains a subpopulation of cells known as cancer stem cells (CSCs). The CSCs are believed to be responsible for chemotherapeutic resistance and are also involved in tumor initiation, progression, evolution, and metastasis to distant sites. The present study aimed to investigate the anti-CSC potential of selected Ethiopian medicinal plants traditionally used for breast cancer treatment., Methods: The solvent fractions of three medicinal plants (the ethyl acetate fraction of Vernonia leopoldi, the aqueous fraction of Sideroxylon oxyacanthum, and the chloroform fraction of Clematis simensis) resulting from the methanolic crude extracts were selected based on their previously demonstrated cytotoxic effects on breast cancer cell lines. The effect of these solvent fractions on the status of the cancer stem cell subpopulation of the JIMT-1 cell line was assessed by flow cytometric evaluation of the proportion of aldehyde dehydrogenase positive cells and by measuring colony forming efficiency in a serum-free soft agar assay after treatment. Effects on cell migration using a wound healing assay and on tumor necrosis factor-α-induced translocation of nuclear factor-kappa B to the cell nucleus were also investigated., Results: The solvent fractions showed a dose-dependent reduction in the aldehyde dehydrogenase positive subpopulation of JIMT-1 cells. The chloroform fraction of C. simensis (80 μg/mL) completely blocked colony formation of JIMT-1 cells. The wound healing assay showed that all fractions significantly reduced cell migration. The ethyl acetate fraction of V. leopoldi (0.87 μg/mL) significantly inhibited tumor necrosis factor-α-induced nuclear factor-kappa B translocation to the nucleus., Conclusion: The solvent fractions of the medicinal plants showed desirable activities against breast cancer stem cells in the JIMT-1 cell line, which warrants further studies.

The current study was aimed at highlighting the role of blood pancreatic amylase in the regulation of glucose homeostasis and insulin secretion in a porcine model of streptozotocin- (STZ-) induced diabetes and in a rat pancreatic beta-cell line, BRIN-BD11. Blood glucose, plasma insulin, and glucagon levels were measured following a duodenal glucose tolerance test (IDGTT), in four pigs with STZ-induced type 2 diabetes (T2D pigs) and in four pigs with STZ-induced type 1 diabetes (T1D pigs). Four intact pigs were used as the control group. The effect of amylase supplementation on both acute and chronic insulin secretion was determined in a BRIN-BD11 cell line. The amylase infusion had no effect on the glucose utilization curve or glucagon levels in the healthy pigs. However, a significant lowering of insulin release was observed in healthy pigs treated with amylase. In the T2D pigs, the glucose utilization curve was significantly lowered in the presence of amylase, while the insulin response curve remained unchanged. Amylase also significantly increased glucagon release during the IDGTT in the T2D and T1D pigs, by between 2- and 4-fold. Amylase did not affect the glucose utilization curve in the T1D pigs. Amylase supplementation significantly decreased both acute and chronic insulin secretion in the BRIN-BD11 cells. These data confirm our previous observations and demonstrate the participation of pancreatic amylase in glucose absorption/utilization. Moreover, the present study clearly highlights the direct impact of pancreatic blood amylase on insulin secretion from pancreatic beta-cells and its interactions with insulin and glucagon secretion in a porcine model., Competing Interests: The authors of this manuscript have the following competing interests: Stefan Pierzynowski is the owner of Anara AB, Sweden, and Vitananano Sp. z o.o./PROF, Poland. Kateryna Pierzynowska is employed by Anara AB, Sweden. SO declares no competing interests., (Copyright © 2020 Kateryna Pierzynowska et al.)