Etoposide Concentrations Research Articles

Feeling Fragile: Chemosensitization of Metastatic Breast Cancer Cells Through the Use of 4EGI‐1 and Etoposide

Breast cancer is the most common cancer among women worldwide. High quantities of Etoposide (ETP), a topoisomerase II inhibitor, have been shown to have considerable adverse effects, therefore making chemosensitization an appropriate area to research. Combination therapies with the drug have been largely unexplored. In the past, ETP has been used to treat metastatic breast cancer in conjunction with other drugs, specifically chemosensitizers. Combining ETP with another drug as a chemosensitizer could increase its efficacy. In past studies, non‐small cell lung cancer has been treated with a novel drug compound, 4EGI‐1, which is a translation inhibitor. Combining the drugs by using 4EGI‐1 to treat metastatic breast cancer as a chemosensitizer could decrease the amount of ETP needed as well as increasing its efficacy. Here we examine the relationship between different concentrations of ETP and 4EGI‐1 and their effect on immobilized breast cancer cell culture. Cell death was used as an assay to determine the efficacy of the drug combination. We hope to show the synergistic effects of ETP and 4EGI‐1 on breast cancer cells. The cell line used was MDA‐MB‐231, a human epithelial breast cancer cell, derived from a 51‐year‐old female diagnosed with mammary adenocarcinoma. The goal of our in vitro studies is to eventually increase patient comfort in a clinical setting by reducing the necessary amount of ETP.Support or Funding InformationUniversity of Minnesota, The Nueva School

Extracellular vesicles from senescent hepatic stellate cells promote cell viability of hepatoma cells through increasing EGF secretion from differentiated THP-1 cells.

Since the discovery of the senescence-associated secretory phenotype, the role of senescent hepatic stellate cells (HSCs) in hepatocellular carcinoma (HCC) development has gained increasing attention. Similar to cytokines, extracellular vesicles (EVs) are essential for intercellular communication. However, the function of EVs derived from senescent HSCs in HCC progression has not been extensively studied. The aims of the present study were to characterize the EVs derived from senescent HSCs and determine their role in the tumor microenvironment. Cellular senescence was induced in human hepatic stellate cells (HHSteCs) with various concentrations of etoposide. Induction was confirmed using EdU staining and 53BP1 and p21 immunostaining. EVs were isolated by ultracentrifugation and analyzed by nanoparticle tracking analysis. Multiplex immunoassays were used to compare the levels of growth factors secreted from hepatoma cell lines and macrophage cells pretreated with EVs derived from senescent HHSteCs (senescent EVs) with those pretreated with EVs derived from normal cultured HHSteCs (normal EVs). Treatment with 25 µM etoposide for 3 days was the most effective at inducing senescence in HHSteCs. This finding was confirmed by induction of irreversible cell-cycle arrest, upregulation of 53BP1 and p21 expression, and increased SA-β-gal staining. Senescent HHSteCs released increased quantities of EV particles compared with normally cultured HHSteCs. Multiplex analysis revealed that there was no difference between hepatoma cell lines treated with normal EVs and those treated with senescent EVs in growth factor secretion. In contrast, the secretion of epidermal growth factor (EGF) was increased by macrophage cells treated with senescent EVs compared with those treated with normal EVs. Furthermore, senescent EVs did not affect the viability of hepatoma cells but increased the viability of hepatoma cells co-cultured with macrophage cells. In conclusion, the release of EVs from senescent HSCs was higher compared with normal HSCs. Furthermore, senescent EVs promoted HCC development by upregulating EGF secretion from macrophages.

In vitro compatibility and stability of admixtures containing etoposide, epirubicin hydrochloride and vindesine sulphate in a single infusion bag.

The etoposide, doxorubicin hydrochloride, vincristine sulphate, cyclophosphamide and prednisone (EPOCH) chemotherapy regimen is effective in patients with relapsed or refractory non-Hodgkin's lymphoma. However, vincristine and doxorubicin hydrochloride are relatively toxic, leading to neurovirulence and cardiotoxicity, respectively. In this study, we replaced these drugs with vindesine and epirubicin hydrochloride to reduce the cardiotoxicity and evaluated admixtures containing these drugs along with etoposide in a single infusion bag in vitro. The appearance and pH of the admixtures were evaluated, and the number of particles was detected. High-performance liquid chromatography was used to measure the concentration and degradation rates of etoposide, epirubicin hydrochloride and vindesine sulphate in each admixture. No precipitation occurred when mixing clinically relevant concentrations of etoposide, epirubicin hydrochloride and vindesine sulphate in a 0.9% NaCl injection solution. Furthermore, the delta pH of the admixtures was ≤0.12 throughout the experiment, and the number of particles (≥10 and ≥25μm) in the solutions over the 24hours post-preparation period met USP standards. Etoposide, epirubicin hydrochloride and vindesine sulphate were retained at >96% of their initial concentrations in the admixtures at 25°C over the course of the experiment. Etoposide, epirubicin hydrochloride and vindesine sulphate are compatible when mixed in a 0.9% NaCl injection solution, and the admixtures are stable for at least 24hours when stored in infusion bags. This in vitro analysis indicates the suitability of our novel admixtures containing less toxic drug equivalents in a single infusion bag for clinical application.

Comparison of ocular pharmacokinetics of etoposide and its nanoemulsion after subtenon administration in rabbits

Background Subtenon anticancer drugs are given as an adjunct to systemic chemotherapy for conditions like retinoblatoma. This study evaluated the ocular kinetics of nano-emulsion formulation of etoposide (NanoEt) and compared it with an equal dose of commercially available alcohol-based etoposide formulation in healthy rabbits. Methods A nanoemulsion formulation of NanoEt was developed and then evaluated for its ocular kinetics by subtenon administration in healthy rabbits. After the sterile subtenon administration of the drug, the eyes were enucleated after CO2 euthanasia at time intervals of 2 h, 6 h, 12 h, and 24 h, and ocular tissues, blood, and plasma were separated. The concentration of etoposide in the ocular tissues and blood was quantified using liquid chromatography tandem mass spectrometry (LC MS/MS). Results This study found that subtenon injection of NanoEt showed 24 times higher concentration in rabbit retina compared to an equal dose of conventional marketed formulation. Based on the ocular tissue bioavailability calculations (AUC0-24), the present study revealed that the formulation enhanced 90% ocular bioavailability of etoposide, when it was injected in the form of nano-emulsion in most of the tissues. Conclusions NanoEt has better bioavailability compared to the commercial alcohol-based formulation for subtenon injection. Low systemic exposure showed further advantage for its projected use in retinoblastoma (Rb) as an adjunct therapy. Further studies in Rb animal models are required to evaluate its safety and efficacy, for its clinical utility.

Etoposide. Documentation of proposed values of occupational exposure limits (OELs)

Etoposide. Documentation of proposed values of occupational exposure limits (OELs)

Deazaflavin Inhibitors of TDP2 with Cellular Activity Can Affect Etoposide Influx and/or Efflux

Tyrosyl DNA phosphodiesterase 2 (TDP2) facilitates the repair of topoisomerase II (TOP2)-linked DNA double-strand breaks and, as a consequence, is required for cellular resistance to TOP2 "poisons". Recently, a deazaflavin series of compounds were identified as potent inhibitors of TDP2, in vitro. Here, however, we show that while somedeazaflavins can induce cellular sensitivity to the TOP2 poison etoposide, they do so independently of TDP2 status. Consistent with this, both the cellular level of etoposide-induced TOP2cleavage complexes and the intracellular concentration of etoposide was increased by incubation with deazaflavin, suggesting an impact of these compounds on etoposide uptake/efflux. In addition, deazaflavin failed to increase the level of TOP2 cleavage complexes or sensitivity induced by m-AMSA, which is a different class of TOP2 poison to which TDP2-defective cells are also sensitive. In conclusion, while deazaflavins are potent inhibitors of TDP2 in vitro, their limited cell permeability and likely interference with etoposide influx/efflux limits their utility in cells.

Optimization by design of etoposide loaded solid lipid nanoparticles for ocular delivery: Characterization, pharmacokinetic and deposition study

The present study was to develop etoposide loaded solid lipid nanoparticles (SLN) and optimize it for effective ocular delivery to the posterior eye. SLN were prepared by melt-emulsification and ultrasonication technique. Etoposide loaded SLN were optimized by using three-factor three levels Box-Behnken design to establish the functional relationships between variables on responses of particle size, polydispersity index (PDI) and entrapment efficiency (EE). SLN were characterized for size & surface morphology, entrapment efficiency and in vitro release. Further the pharmacokinetic study of optimized formulation after intravitreal administration was evaluated in Wister rats. The deposition in the ocular tissues was checked by scintigraphic analysis in Albino rabbits. Histology was also done to evaluate morphological changes if any occur after treatment. The particle size, PDI and EE obtained for the optimized formulation (Z15) were 239.43 ± 2.35 nm, 0.261 ± 0.001 and 80.96 ± 2.21% respectively. Single intravitreal administrations of SLN were able to give sustained etoposide concentration in the vitreous for 7 consecutive days which was also supported by the results of Gamma scintigraphic study. Histology of posterior ocular tissues do not showed any serious toxic effect. Therefore it can concluded that etoposide loaded SLN was able to maintain vitreous concentration of drug without any serious toxic effect to the surrounding ocular tissues after an intravitreous administration in rat eye.

A Population Dynamic Energy Budget-Based Tumor Growth Inhibition Model for Etoposide Effects on Wistar Rats.

This work aimed to develop a population PK/PD tumor-in-host model able to describe etoposide effects on both tumor cells and host in Walker-256 tumor-bearing rats. Etoposide was investigated on thirty-eight Wistar rats randomized in five arms: two groups of tumor-free animals receiving either placebo or etoposide (10mg/kg bolus for 4days) and three groups of tumor-bearing animals receiving either placebo or etoposide (5 or 10mg/kg bolus for 8 or 4days, respectively). To analyze experimental data, a tumor-in-host growth inhibition (TGI) model, based on the Dynamic Energy Budget (DEB) theory, was developed. Total plasma and free-interstitial tumor etoposide concentrations were assessed as driver of tumor kinetics. The model simultaneously describes tumor and host growths, etoposide antitumor effect as well as cachexia phenomena related to both the tumor and the drug treatment. The schedule-dependent inhibitory effect of etoposide is also well captured when the intratumoral drug concentration is considered as the driver of the tumor kinetics. The DEB-based TGI model capabilities, up to now assessed only in mice, are fully confirmed in this study involving rats. Results suggest that well designed experiments combined with a mechanistic modeling approach could be extremely useful to understand drug effects and to describe all the dynamics characterizing in vivo tumor growth studies.

Selective Electrochemical Determination of Etoposide Using a Molecularly Imprinted Overoxidized Polypyrrole Coated Glassy Carbon Electrode

A simple and efficient new electrochemical sensor based on molecularly imprinted polymer has been developed for selective detection of an anticancer agent Etoposide (ETP). The sensor was prepared by electropolymerization via cyclic voltammetry (CV) of pyrrole onto a glassy carbon electrode (GCE) in the presence of ETP molecules. The extraction of ETP molecules embedded in the polymeric matrix was carried out by overoxidation in sodium hydroxide medium using CV. Various important parameters affecting the performance of the imprinted film (MIP) coated sensor were studied and optimized using differential pulse voltammetry (DPV). Under optimal conditions, the sensor response exhibited a linear dependence on ETP concentration (R2= 0.999) over the range 5.0×10−7M – 1.0×10−5M with a LOD (3σ/m) of 2.8×10−9M. The precision (% RSD, n=6) of the proposed sensor for intra- and interdays was found to be 0.84 and 2.46%, respectively. The selectivity of MIP/GCE sensor toward ETP was investigated in the presence of different interfering molecules including excipients and ETP metabolites. The developed sensor showed great recognition ability toward ETP and was successfully applied for its determination in injectable dosage forms and biological human fluids.

Lowering Etoposide Doses Shifts Cell Demise From Caspase-Dependent to Differentiation and Caspase-3-Independent Apoptosis via DNA Damage Response, Inducing AML Culture Extinction.

Cytotoxic chemotherapy, still the most widely adopted anticancer treatment, aims at eliminating cancer cells inducing apoptosis with DNA damaging agents, exploiting the differential replication rate of cancer vs. normal cells; efficiency is evaluated in terms of extent of induced apoptosis, which depends on the individual cell sensitivity to a given drug, and on the dose. In this in vitro study, we report that the concentration of etoposide, a topoisomerase II poison widely used in clinics, determines both the kinetics of cell death, and the type of apoptosis induced. We observed that on a set of myeloid leukemia cell lines, etoposide at high (50 uM) dose promoted a rapid caspase-3-mediated apoptosis, whereas at low (0.5 uM) dose, it induced morphological and functional granulocytic differentiation and caspase-2-dependent, but caspase-3-independent, cell death, displaying features consistent with apoptosis. Both differentiation and caspase-2- (but not 3)-mediated apoptosis were contrasted by caffeine, a well-known inhibitor of the cellular DNA damage response (DDR), which maintained cell viability and cycling, indicating that the effects of low etoposide dose are not the immediate consequence of damage, but the result of a signaling pathway. DDR may be thus the mediator responsible for translating a mere dosage-effect into different signal transduction pathways, highlighting a strategic action in regulating timing and mode of cell death according to the severity of induced damage. The evidence of different molecular pathways induced by high vs. low drug doses may possibly contribute to explain the different effects of cytotoxic vs. metronomic therapy, the latter achieving durable clinical responses by treating cancer patients with stable, low doses of otherwise canonical cytotoxic drugs; intriguingly caspase-3, a major promoter of wounded tissue regeneration, is also a key factor of post-therapy cancer repopulation. All this suggests that cancer control in response to cytotoxic drugs arises from complex reprogramming mechanisms in tumor tissue, recently described as anakoinosis.

Pharmacokinetic interaction between mitotane and etoposide in adrenal carcinoma: a pilot study.

BackgroundThe combination of mitotane and platinum-etoposide chemotherapy is a front-line treatment in metastatic adrenocortical carcinoma (ACC), although this regimen shows limited efficacy. Pharmacokinetic drug–drug interaction between mitotane, a strong CYP3A4 inducer, and etoposide, which is a substrate of CYP3A4, may contribute to chemoresistance. The aim of this pilot study was to assess the pharmacokinetic interaction between mitotane and etoposide in ACC patients.MethodsFive consecutive ACC patients treated with platinum etoposide (120–150 mg/m2 day 1–2–3 at cycle 1), with or without concomitant mitotane, were included. In the absence of limiting toxicity, a dose escalation of etoposide was proposed since cycle 2. Plasma etoposide concentrations were measured using liquid chromatography at 0, 4 and 24 h after each infusion. Clearance and area under the curve (AUC) of etoposide were determined at each cycle.ResultsPatients received two to six chemotherapy cycles, in association with mitotane (N = 4) or after mitotane discontinuation (N = 1). Etoposide clearance was two-fold higher with concomitant mitotane (4.95 L/h) than after mitotane discontinuation (2.53 L/h, P = 0.014), and 2.5-fold higher than that in reference population not treated with mitotane (1.81 L/h). Etoposide dose escalation was performed in four patients under mitotane, resulting in two minor tumor responses and one severe toxicity (febrile aplasia) at dose of 300 mg/m2/day. Tumor response was associated with higher etoposide AUC (267.3 vs 188.8 mg.h/L, P = 0.04).ConclusionA drug–drug interaction between mitotane and etoposide may contribute to the low efficacy of platinum-etoposide chemotherapy. This pilot study suggests further a potential benefit of increasing etoposide dose in ACC patients receiving mitotane.

Differentiated and exponentially growing HL60 cells exhibit different sensitivity to some genotoxic agents in the comet assay

The aim of this study was to investigate the effect of the cell differentiation status on the sensitivity to genotoxic insults. For this, we utilized the comet assay to test the DNA damage after treatment with 5 different substances with different mechanism of action in human promyelocytic HL60 cells with or without cell differentiation.A 4-hour MMS treatment induced a significant and concentration-dependent increase in DNA damage for both differentiated and undifferentiated cells, but the difference in sensitivity was only significant at the highest concentration. A 4-hour doxorubicin treatment did not induce DNA damage in differentiated HL60 cells, while it did in undifferentiated cells with its highest tested concentration. A one-hour etoposide treatment caused significant increase in DNA damage concentration dependently in both cell variants. This DNA damage was significantly higher in undifferentiated HL60 cells with several tested concentrations of etoposide. The treatment with the oxidizing substances hydrogen peroxide and potassium bromate yielded significant DNA damage induction in both undifferentiated and differentiated cells with no difference according to the differentiation status.Doxorubicin and etoposide are known to inhibit topoisomerase II. The activity of this enzyme has been shown to be higher in undifferentiated actively proliferating cells than in differentiated cells. This may be of relevance when exposures to topoisomerase-inhibiting compounds or the genotoxicity of compounds with unknown mechanism of action are assessed in routine testing.

Etoposide-resistance in a neuroblastoma model cell line is associated with 13q14.3 mono-allelic deletion and miRNA-15a/16-1 down-regulation

Drug resistance is the major obstacle in successfully treating high-risk neuroblastoma. The aim of this study was to investigate the basis of etoposide-resistance in neuroblastoma. To this end, a MYCN-amplified neuroblastoma cell line (HTLA-230) was treated with increasing etoposide concentrations and an etoposide-resistant cell line (HTLA-ER) was obtained. HTLA-ER cells, following etoposide exposure, evaded apoptosis by altering Bax/Bcl2 ratio. While both cell populations shared a homozygous TP53 mutation encoding a partially-functioning protein, a mono-allelic deletion of 13q14.3 locus, where the P53 inducible miRNAs 15a/16-1 are located, and the consequent miRNA down-regulation were detected only in HTLA-ER cells. This event correlated with BMI-1 oncoprotein up-regulation which caused a decrease in p16 tumor suppressor content and a metabolic adaptation of HTLA-ER cells. These results, taken collectively, highlight the role of miRNAs 15a/16-1 as markers of chemoresistance.

Mifepristone potentiates etoposide toxicity in Hep G2 cells by modulating drug transport

Etoposide is a well-known and widely used anticancer drug that displays several side effects. In addition, tumors often acquire resistance to this drug. Our aim is to develop a combination therapy that would augment toxicity of etoposide in malignant cells. Based on literature and our experiments, we selected mifepristone (RU486) as a potential supporting molecule that is able to enhance etoposide toxicity against cancer cells. All experiments were performed with Hep G2 cells, a well-known and described human hepatocellular carcinoma cell line. By using xCELLigence system, we demonstrated that mifepristone enhances toxicity of etoposide in a dose dependent manner with concomitant caspase-3 activity. We evaluated upregulation of Bax because mifepristone was demonstrated to modulate proapoptotic Bax protein expression. Our data show only weak and not statistically significant increase of Bax expression. On the other hand, we show that mifepristone increases etoposide toxicity via inhibition of ABC transporters, coupled with significant increase of intracellular etoposide concentration. In conclusion, we demonstrate that mifepristone has a synergistic effect with etoposide treatment in the Hep G2 cells and that the effect is related to ABC transporters inhibition.

Comparison of different immunoassays for γH2AX quantification

Background: One of the most sensitive methods for DNA double-strand break (DSB) detection is the assessment of phosphorylated histone protein 2AX (γH2AX), which can be visualized as γH2AX focus surrounding the damage site. Quantification of γH2AX offers a variety of potential diagnostic applications being on the brink of introduction into clinical routine. Therefore, further assay optimization and automation is required to enable standardized, high-throughput γH2AX analysis. To quantify γH2AX levels, different immunoassay techniques can be performed, each showing specific characteristics regarding assay sensitivity and sample processing. Although microscopic foci quantification is considered the most sensitive approach, data of direct comparison of multiple γH2AX immunoassay techniques are rare. In the current study we compared γH2AX quantification by different methods, including automated fluorescence microscopy, flow cytometry as well as immunoblotting. Further, we discussed assay-specific advantages and disadvantages. Methods: Isolated human peripheral blood mononuclear cells (PBMCs) were exposed to various concentrations of the DNA DSB-inducing, cytostatic drug etoposide for one hour. Subsequently, γH2AX levels were assessed by flow cytometry, immunoblotting and automated microscopy. Results: Automated fluorescence microscopic foci quantification revealed the lowest limit of detection (LoD) (0.53 µM etoposide). More than 10-fold higher etoposide concentrations were required to distinguish γH2AX values form background signal by fluorescence intensity-based methods like flow cytometry. Immunoblotting showed the poorest LoD of all three techniques. Conclusions: In contrast to flow cytometry and immunoblotting, automated fluorescence γH2AX foci quantification showed the lowest LoD. This low LoD allows the assessment and follow-up of patients with respective antitumor therapy. Thus, to apply the most suitable γH2AX analysis, specific assay characteristics must be considered.

Toxicogenomic responses of low level anticancer drug exposures in Daphnia magna

The use of anticancer drugs in chemotherapy is increasing, leading to growing environmental concentrations of imatinib mesylate (IMA), cisplatinum (CDDP), and etoposide (ETP) in aquatic systems. Previous studies have shown that these anticancer drugs cause DNA damage in the crustacean Daphnia magna at low, environmentally relevant concentrations. To explore the mechanism of action of these compounds and the downstream effects of DNA damage on D. magna growth and development at a sensitive life stage, we exposed neonates to low level concentrations equivalent to those that elicit DNA damage (IMA: 2000 ng/L, ETP: 300 ng/L, CDDP: 10 ng/L) and performed transcriptomic analysis using an RNA-seq approach. RNA sequencing generated 14 million reads per sample, which were aligned to the D. magna genome and assembled, producing approximately 23,000 transcripts per sample. Over 90% of the transcripts showed homology to proteins in GenBank, revealing a high quality transcriptome assembly, although functional annotation was much lower. RT-qPCR was used to identify robust biomarkers and confirmed the downregulation of an angiotensin converting enzyme-like gene (ance) involved in neuropeptide regulation across all three anticancer drugs and the down-regulation of DNA topoisomerase II by ETP. RNA-seq analysis also allowed for an in depth exploration of the differential splicing of transcripts revealing that regulation of different gene isoforms predicts potential impacts on translation and protein expression, providing a more meaningful assessment of transcriptomic data. Enrichment analysis and investigation of affected biological processes suggested that the DNA damage caused by ETP and IMA influences cell cycle regulation and GPCR signaling. This dysregulation is likely responsible for effects to neurological system processes and development, and overall growth and development. Our transcriptomic approach provided insight into the mechanisms that respond to DNA damage caused by anticancer drug exposure and generated novel hypotheses on how these chemicals may impact the growth and survival of this ecologically important zooplankton species.

Abstract 1565: Identification of genetic and epigenetic biomarkers for prediction of chemoresistance in the treatment of high-risk advanced retinoblastoma

Abstract Background: Retinoblastoma (RB) is the most common pediatric retinal tumor initiated by biallelic inactivation of the retinoblastoma gene (RB1). Currently, chemotherapy with carboplatin, vincristine, and etoposide followed by optic enucleation is the most commonly used for retinoblastoma patients in China. The recurrence and metastasis of high-risk advanced RB is still a major obstacle for successful chemotherapy with very low survival rate only 26% in China, mainly due to chemotherapeutic resistance. The aim of the study is to identify genetic/epigenetic biomarkers with regarding to the prediction of outcome of chemotherapy for high-risk advanced RB, and to functionally characterize for mechanisms of candidate genes or potential biomarkers. Methods: Total of 41 pediatric patients with D- or E-stage retinoblastoma according to International Intraocular Retinoblastoma Classification (IIRC) staging standard were collected in the present study. Four complete remission (CR) and four recurrence patients due to their typical symptoms were selected to compare differences in profiles of whole-exome and genomic DNA methylation with high throughput next-generation sequencing technology by using their peripheral blood samples and to identify candidates in an integrative analysis of omics datasets. Subsequently, functional validation study was performed by using a human retinoblastoma Y79 cell line for those relevant candidate genes. Specifically, Y79 cells were knockdown or overexpressed with specific siRNA or expression vectors, respectively, followed by treatments with a diluted concentrations of carboplatin, vincristine, and etoposide in Cell Counting Kit (CCK-8) assay. Results: There were significantly different profiles of whole-exome and genomic DNA methylation in the integrative analysis. Total of 556 SNPs and 35 Indels were discriminated from whole-exome sequencing data, and 721 DNA methylation sites were detected with significant differences in RRBS assay between the datasets of those recurrence and CR patients. One variant (rs140693) in the exon 3 of MBD4 gene was identified in all those 4 recurrence patients and down-regulation of this gene by its specific siRNAs significantly altered the resistance of Y79 cells to all those above three drugs, as well as the cell growth rate, suggesting that the candidate variant/gene might be functional to impact on multiple drug resistances to carboplatin, vincristine and etoposide. Further functional studies would be required for characterization of its action mechanisms. Conclusion: Our study had identified novel genetic/epigenetic biomarkers to predict resistance against chemotherapeutics for high-risk advanced RB. It would be helpful as the first step toward precise medicine to explore intrinsic mechanism of chemoresistance and develop potential therapeutic targets for RB in future. Citation Format: Wenping Song, Rongguang Shao, Liang Li. Identification of genetic and epigenetic biomarkers for prediction of chemoresistance in the treatment of high-risk advanced retinoblastoma [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 1565.

PO-093 A hep-2 cell model as tool for analysing DNA double strand breaks in cancer patients

IntroductionDNA double strand breaks (DSB) are one of the most severe DNA damages and as thus can lead to genomic instability favouring cancer. The number of phosphorylated histone variant H2AX (γH2AX) correlates with the number of DSB and can be quantitatively detected by immunofluorescence imaging. In particular, we are interested on the individual response and adverse effects of DSB–inducing chemotherapeutic drugs such as etoposide in the context of the chemoresistance–inducing and tumour–associated antigen dense fine speckled protein of 70 kDa (DFS–70).Material and methodsHEp-2 wild type and CRISRP/Cas9-generated DFS-70 knock-out (DFS-70 KO) cells were treated with the topoisomerase inhibitor etoposide (0–10 µM) to induce DSB. The AKLIDES NUK system (Medipan GmbH, Dahlewitz, Germany), a fully automated and standardised immunofluorescence imaging and data processing platform, was used to measure the number of γH2AX foci and nucleus size. Additionally, immunoblotting was performed to analyse yH2AX.Results and discussionsBoth the protein analysis by Western blotting and Immunofluorescence analysis by AKLIDES NUK system showed an elevated formation of γH2AX with increased etoposide concentration. HEp-2 wild type cells indicated a significant induction (p<0.01) of DSB up to 13 foci per cell after 16 hours of etoposide treatment. Adverse effects could be observed on nucleus size that increased significantly upon treatment in all cell lines. No difference in the basal level of γH2AX foci were detectable comparing wild type and DSF-70 KO cells. In contrast, two out of five DFS-70 KO clones exhibited an increased foci number in comparison to the wild type control after 16 hours of etoposide treatment. This finding might indicate that DSF-70 KO sublines are more sensitive to topoisomerase inhibition than HEp–2 wild type cells.ConclusionThe automated multiparameter imaging platform AKLIDES allows an individual and differential detection of γH2AX foci formation and nuclei size in human laryngeal carcinoma cells, HEp–2 and sublines. Therefore, this system can be useful to identify cancer progression e.g. analysing peripheral blood mononuclear cells of patients with malignant lymphoma. Moreover, additional biomarkers defining cancer progression can be integrated into the analysis.

Etoposide – inhalable fraction. Determination method in workplace air

Etoposide at room temperature is a fine white to yellow-brown crystalline odorless powder. Etoposide is one of the most widely used cytotoxic drugs and has strong antitumour activity in cases of small-cell lung cancer, testicular cancer or lymphoma. Occupational exposure to etoposide (mainly via skin contact or via inhalation route) may occur among group of healthcare workers or workers employed in the production of this drug. Exposure to etoposide can cause suppression of bone marrow function and gastrointestinal symptoms such as nausea, vomiting, bronchospasm, inflammation of mucous membranes, hair loss and secondary leukemia. Agency for Research on Cancer (IARC) has classified etoposide as a compound probably carcinogenic to humans (Group 2.A) and in combination with cisplatin and bleomycin as carcinogenic to humans (Group 1). The aim of this study was to develop and validate a sensitive method for determining inhalable fraction of etoposide concentrations in workplace air in the range from 1/10 to 2 MAC values, in accordance with the requirements of Standard PN-EN 482. The study was performed using a liquid chromatograph with tandem mass detection (HPLC-MS/MS). All chromatographic analysis were perfomed with Supelcosil LC 18 150 × 3 mm analytical column, which was eluted with a mixture of methanol and water with 0.1% of formic acid. This method is based on collecting inhalable fraction of etoposide on glass fiber filter, extracting with a mixture of methanol: water with addition of formic acid (0.1%), and chromatographic determining of resulted solution with HPLC-MS/MS technique. The average extraction efficiency of etoposide from filters was 90%. The method is linear (r = 0.9985) within the investigated working range from 0.036 μg/ml to 1.44 μg/ml. The calculated limit of detection (LOD) and the limit of quantification (LOQ) were 0.0086 and 0.0026 μg/ml, respectively. The analytical method described in this paper, thanks to HPLC MS/MS technique, enables specific and selective determination of inhalable fraction of etoposide in workplace air in the presence of other compounds at concentrations from 0.0001 mg/m3 (1/20 proposed MAC value). The method is precise, accurate and it meets the criteria for measuring chemical agents listed in Standard No. EN 482. The method can be used for assessing occupational exposure to etoposide and associated risk to workers’ health. The developed method of determining etoposide has been recorded as an analytical procedure (see appendix).

Effect of etoposide-induced alteration of the Mdm2-Rb signaling pathway on cellular senescence in A549 lung adenocarcinoma cells.

The present study aimed to investigate the effect of various concentrations of etoposide (VP-16) on the E3 ubiquitin-protein ligase Mdm2 (Mdm2)-retinoblastoma (Rb) signaling pathway in the cellular senescence of A549 lung adenocarcinoma cells. A549 cells were randomly divided into the following four groups: Control group (no treatment), group 1 (1 µmol/l VP-16), group 2 (5 µmol/l VP-16) and group 3 (25 µmol/l VP-16). Each group was cultured for 48 h after treatment prior to observation of the alterations to cellular morphology. The cell cycle distribution of each group was also detected by flow cytometry. In addition, the activity of cellular senescence-associated β-galactosidase, and the expression of Mdm2 and phosphorylated (p-) Rb protein, was measured. The percentage of senescent cells was significantly higher following VP-16 treatment compared with the control group. The percentage of G1 phase cells, and p-Rb protein and Mdm2 protein expression were also significantly different following VP-16 treatment compared with the control group. VP-16 increased the activity of β-galactosidase in the A459 cells. VP-16 also decreased the expression level of Mdm2 and p-Rb protein and inhibited cell cycle progression in G1. These results indicate that VP-16 induces the cellular senescence of A549 cells via the Mdm2-Rb signaling pathway. However, further investigations are required to validate the mechanisms underlying these effects of VP-16.