Etoposide Concentrations Research Articles

Abstract 7224: Investigation of the efficacy of duocarmycin SA in combination with FDA approved drugs in acute myeloid leukemia cells in vitro

Abstract Acute myeloid leukemia (AML) is a hematological malignancy that is characterized by the arrest of hematopoietic stem cell differentiation, clonal expansion of myeloid hematopoietic precursors and poor patient survival outcomes. Despite therapeutic advancements, the prognosis of AML patients remains poor and improving patient survival outcomes continues to be a challenge. Therefore, the objective of this study was to investigate the efficacy of Duocarmycin SA (DSA- an antitumor antibiotic that induces DNA alkylation) in combination with FDA approved drugs currently used in the treatment of AML. In this study, DSA was evaluated in combination with doxorubicin (an anthracycline that disrupts topoisomerase II mediated DNA repair) or etoposide (a topoisomerase II inhibitor used to treat relapsed/refractory AML) in human AML cells in vitro. We hypothesized that DSA in combination with doxorubicin or etoposide would exhibit synergistic effects by improving the efficacy of both doxorubicin and etoposide in AML cells. The human AML cell lines (Molm-14 and HL60) were used for all studies. The phenotype of the cells was confirmed by flow cytometry and the IC50 of the drugs were determined using the MTT assay. Our results showed that the AML cells were CD45+, CD33+, CD13+ and CD4+. The IC50 of DSA alone for Molm-14 cells and HL60 cells were 11.12 pM and 114.8 pM, respectively. DSA alone induced DNA double-stranded breaks, significantly decreased the production of colonies and significantly increased apoptosis in AML cells in a dose-dependent manner. The IC50 of doxorubicin alone and etoposide alone was 68.6 nM and 129.7 nM, respectively. DSA in combination with the FDA approved AML drugs showed increased cytotoxic efficacy by observable decreases in AML cell viability. In summary, 1) picomolar concentrations of DSA alone was sufficient to significantly reduce AML cell viability; 2) DSA in combination with doxorubicin or etoposide significantly reduced AML cell viability and reduced the IC50 concentration of doxorubicin and etoposide. Thus, highlighting the therapeutic potential of using DSA in combination with doxorubicin or etoposide as a drug combination strategy to improve AML patient survival outcomes by decreasing the concentration of drugs necessary to treat patients and reducing off-target toxicity. Citation Format: William A. Chen, Diego Aguilar, Leena So, Yasmeen Jawhar, Nhi Nguyen, Natalie Drew, Terry G. Williams, Carlos A. Casiano, Sinisa Dovat, Kristopher Boyle, Olivia L. Francis-Boyle. Investigation of the efficacy of duocarmycin SA in combination with FDA approved drugs in acute myeloid leukemia cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7224.

Comprehensive analysis of exosome gene LYPD3 and prognosis/immune cell infiltration in lung cancer.

Lung cancer (LC) is a leading cause of cancer-associated mortality worldwide, with high incidence and mortality rates. Ly6/PLAUR domain containing 3 (LYPD3) is a tumorigenic and highly glycosylated cell surface protein that has been rarely reported in LC. This study aimed to explore the prognostic role and immune cell infiltration of LYPD3 in LC. We used ExoCarta, a database of exosomal proteins and RNA, to select exosomes in LC. The Tumor Immune Estimation Resource (TIMER) and Human Protein Atlas (HPA) databases were utilized to compare the expression of LYPD3 in LC. We applied Gene Expression Profiling Interactive Analysis 2 (GEPIA2) and Kaplan-Meier (KM) plotter to evaluate the prognostic prediction performance of LYPD3. Biological processes (BPs), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, and gene set enrichment analysis (GSEA) analyses were performed to illustrate the possible role of LYPD3 in LC. The correlations between LYPD3 and immune cell infiltration were explored using Tumor and Immune System Interaction Database (TISIDB), GEPIA2, and TIMER. R software was used for statistical analysis and mapping. A total of 904 exosome molecules were screened in LC. Further analysis showed that the up-regulation of LYPD3 in these 904 exosome molecules was associated with poor prognosis in LC. Pan-cancer analyses revealed that the expression of LYPD3 varied in many cancers, particularly in LC. Clinical correlation analysis indicated that LYPD3 was associated with stage and T classification in LC. We observed that LYPD3 co-expression genes were associated with cell cycle, DNA replication, proteasome, and regulation of the actin cytoskeleton by GSEA. Moreover, LYPD3 was associated with immune modulators. Immunophenoscores (IPS) and IPS-CTLA4 were significantly different between the high LYPD3 group and low LYPD3 group. Additionally, the median half maximal inhibitory concentration (IC50) of bexarotene, cyclopamine, etoposide, and paclitaxel in LYPD3 high group was significantly lower than that in LYPD3 low group. LYPD3 is involved in many BPs of LC, such as regulating immune cell infiltration and affecting prognosis. Therefore, LYPD3 may have potential value as a biomarker for prognosis and immunotherapy in LC.

Rapid Detection of the Anti-Tumor Drug Etoposide in Biological Samples by Using a Nanoporous-Gold-Based Electrochemical Sensor.

Monitoring etoposide is important due to its wide usage in anti-tumor therapy; however, the commonly used HPLC method is expensive and often requires complicated extraction and detection procedures. Electrochemical analysis has great application prospects because of its rapid response and high specificity, sensitivity, and efficiency with low cost and high convenience. In this study, we constructed a nanoporous gold (NPG)-modified GCE for the detection of etoposide. The electrochemical oxidation of etoposide by NPG caused a sensitive current peak at +0.27 V with good reproductivity in 50 mM of phosphate buffer (pH 7.4). The relationship between etoposide concentration and peak current was linear in the range between 0.1 and 20 μM and between 20 and 150 μM, with a detection sensitivity of 681.8 μA mM-1 cm-2 and 197.2 μA mM-1 cm-2, respectively, and a limit of detection (LOD) reaching 20 nM. The electrode had a good anti-interference ability to several common anions and cations. Spiked recovery tests in serum, urine, and fermentation broth verified the excellent performance of the sensor in terms of sensitivity, reproducibility, and specificity. This may provide a promising tool for the detection of etoposide in biological samples.

New Dual Inhibitors of Tyrosyl-DNA Phosphodiesterase 1 and 2 Based on Deoxycholic Acid: Design, Synthesis, Cytotoxicity, and Molecular Modeling.

Deoxycholic acid derivatives containing various heterocyclic functional groups at C-3 on the steroid scaffold were designed and synthesized as promising dual tyrosyl-DNA phosphodiesterase 1 and 2 (TDP1 and TDP2) inhibitors, which are potential targets to potentiate topoisomerase poison antitumor therapy. The methyl esters of DCA derivatives with benzothiazole or benzimidazole moieties at C-3 demonstrated promising inhibitory activity in vitro against TDP1 with IC50 values in the submicromolar range. Furthermore, methyl esters 4d-e, as well as their acid counterparts 3d-e, inhibited the phosphodiesterase activity of both TDP1 and TDP2. The combinations of compounds 3d-e and 4d-e with low-toxic concentrations of antitumor drugs topotecan and etoposide showed significantly greater cytotoxicity than the compounds alone. The docking of the derivatives into the binding sites of TDP1 and TDP2 predicted plausible binding modes of the DCA derivatives.

DNA Damage Response After Treatment of Cycling and Quiescent Cord Blood Hematopoietic Stem Cells With Distinct Genotoxic Noxae.

Hematopoietic stem cells (HSC) from cord blood can be applied as an alternative to bone marrow in transplantation to treat hematological diseases. Umbilical cord blood (UCB) consists of cycling and non-cycling CD34+/CD45low cells needed for long-term and short-term engraftment. After sorting and subsequent in vitro culture, quiescent HSCs enter the cell cycle. This enables the analysis of HSCs in 2 different cell cycle stages and the comparison of their responses to different genotoxic noxae. To analyze different mechanisms of DNA damage induction in cells, 2 different genotoxins were compared: etoposide, a topoisomerase II inhibitor that targets mitosis in the S/G2-phase of the cell cycle and the alkylating nitrosamine N-Nitroso-N-methylurea (MNU), which leads to the formation of methyl DNA adducts resulting in DNA double breaks during DNA replication and persistent mutations. Cycling cells recovered after treatment even with higher concentrations of etoposide (1.5µM/ 5µM/10µM), while sorted cells treated with MNU (0.1mM/0.3mM/0.5mM/1mM/3Mm/ 5mM) recovered after treatment with the lower MNU concentrations whereas high MNU concentrations resulted in apoptosis activation. Quiescent cells were not affected by etoposide treatment showing no damage upon entry into the cell cycle. Treatment with MNU, similarly to the cycling cells, resulted in a dose-dependent cell death. In conclusion, we found that depending on the genotoxic trigger and the cycling status, CD34+cells have distinct responses to DNA damage. Cycling cells employ both DDR and apoptosis mechanisms to prevent damage accumulation. Quiescent cells predominantly undergo apoptosis upon damage, but their cell cycle status protects them from certain genotoxic insults.

DNA-PK inhibition extends the therapeutic effects of Top2 poisoning to non-proliferating cells, increasing activity at a cost

Type II topoisomerase (Top2) poisoning therapy is used to treat a broad range of cancers via induction of double strand breaks (DSBs) in cells undergoing replication and transcription. Preventing the repair of DSBs via inhibition of DNA-PK, an inhibitor of non-homologous end-joining (NHEJ), increases cell kill with Top2 poisons and has led to the initiation of several clinical trials. To elucidate the cellular mechanisms leading to synergistic activity of dual DNA-PK/Top2 inhibition we looked at their effects in cycling versus non-cycling cells, in 3D spheroids and in xenograft models. Combined DNA-PK/Top2 inhibition was found to not only increase the cell kill in proliferating cells, the cell population that is typically most vulnerable to Top2 poisoning, but also in non-proliferative but transcriptionally active cells. This effect was observed in both cancer and normal tissue models, killing more cells than high concentrations of etoposide alone. The combination treatment delayed tumor growth in mice compared to Top2 poisoning alone, but also led to increased toxicity. These findings demonstrate sensitization of Top2β-expressing, non-cycling cells to Top2 poisoning by DNA-PK inhibition. Expansion of the target cell population of Top2 poison treatment to include non-proliferating cells via combination with DNA damage repair inhibitors has implications for efficacy and toxicity of these combinations, including for inhibitors of DNA-PK currently in clinical trial.

Kinetic analysis of p53 gene network with time delays and PIDD

p53 kinetics plays a key role in regulating cell fate. Based on the p53 gene regulatory network composed by the core regulatory factors ATM, Mdm2, Wip1, and PIDD, the effect of the delays in the process of transcription and translation of Mdm2 and Wip1 on the dynamics of p53 is studied theoretically and numerically. The results show that these two time delays can affect the stability of the positive equilibrium. With the increase of delays, the dynamics of p53 presents an oscillating state. Further, we also study the effects of PIDD and chemotherapeutic drug etoposide on the kinetics of p53. The model indicates that (i) PIDD low-level expression does not significantly affect p53 oscillatory behavior, but high-level expression could induce two-phase kinetics of p53; (ii) Too high and too low concentration of etoposide is not conducive to p53 oscillation. These results are in good agreement with experimental findings. Finally, we consider the influence of internal noise on the system through Binomial [Formula: see text]-leap algorithm. Stochastic simulations reveal that high-intensity noise completely destroys p53 dynamics in the deterministic model, whereas low-intensity noise does not alter p53 dynamics. Interestingly, for the stable focus, the internal noise with appropriate intensity can induce quasi-limit cycle oscillations of the system. Our work may provide the useful insights for the development of anticancer therapy.

Abstract 6108: Novel functional dSTRIDE-HR assays to report on the status of homologous recombination repair in cancer cells

Abstract Since the early days of the synthetic lethality concept in DNA Damage Response the status of homologous recombination (HR) repair in cancer cells have been the focus of attention of researchers and clinicians. While different approaches exist, such as the RAD51 immunofluorescence (IF) or HRD genomic assays, functional biomarkers that can assess HR proficiency are missing. We report here the development, optimization and validation of two complementary, HR-specific functional assays. The assays, which are based on the STRIDE platform technology, detect double-strand DNA breaks localized in close proximity to RPA or RAD51 proteins. The optimization phase of assay development was performed in U2OS cells. First, repeatability (intra-run variation) and reproducibility (inter-run variation) of the assays were measured in untreated cells. Then, a series of technical negative controls was performed which have shown that the number of false-positive readouts is below 10% of the total number of signals. Finally, treatment of cells with compounds known to induce double-strand DNA breaks (etoposide and cisplatin) resulted in statistically significant increase in the number of detected dSTRIDE-RAD51 and dSTRIDE-RPA foci when compared to untreated controls. The assays were further validated in NCI-H661 (BRCA2 wild-type) and NCI-H169 (BRCA2 KO) cell line pair. The cells were treated with two concentrations of etoposide and the readouts from dSTRIDE, detecting the total pool of DSBs and dSTRIDE-HR assays were compared. In NCI-H661 cells, treatment with etoposide resulted in an increase in the number of double-strand breaks detected by dSTRIDE and as expected, more DSBs were formed after treatment with the higher concentration. dSTRIDE-HR assays confirmed that approximately 15% and 10% of these DSBs contain RPA and RAD51 proteins, respectively. In NCI-H169 cells etoposide produced a stronger reaction with even more DSBs detected by dSTRIDE, but importantly, no increase in the number of dSTRIDE-RAD51 foci was observed. dSTRIDE-RPA foci increased after treatment hinting that this step of HR remains unperturbed. Interestingly, the number of dSTRIDE-RAD51 foci in untreated cells was comparable between the two cell lines. In conclusion, we show here that two newly developed dSTRIDE-HR assays are well validated and can be successfully applied to report on the status of homologous recombination repair in different cell models. Citation Format: Kamil Solarczyk, Agnieszka Waligórska, Karolina Uznańska, Zsombor Prucsi, Olga Wójcikowska, Ewelina Matuszyk, Magdalena Bartyńska, Agata Kitlińska, Aleksandra Bober, Franek Sierpowski, Maja Białecka, Monika Jarosz, Malgorzata Szczygiel, Szymon Koman, Karolina Korpanty, Lukasz Beben, Lukasz Bandzarewicz, Przemyslaw Stachura, Magdalena Kordon-Kiszala. Novel functional dSTRIDE-HR assays to report on the status of homologous recombination repair in cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6108.

Effect of metformin alone and in combination with etoposide and epirubicin on proliferation, apoptosis, necrosis, and migration of B-CPAP and SW cells as thyroid cancer cell lines.

There has not been a comprehensive study on the simultaneous effects of metformin, etoposide, and epirubicin on thyroid cancer cells. Hence, the current research proposed the in vitro study on the effect of metformin alone and in combination with etoposide and epirubicin on the rate of proliferation, apoptosis, necrosis, and migration against B-CPAP and SW-1736 cells as thyroid cancer cell lines. MTT-based proliferation assay, combination index method, flow cytometry, and scratch wound healing assays were used to evaluate the simultaneous effects of the three approved drugs against thyroid cancer cells. This study showed that the toxic concentration of metformin on normal Hu02 cells was more than 10 folds higher than B-CPAP and SW cancerous cells. Metformin in combination with epirubicin and etoposide could increase percentages of B-CPAP and SW cells in early and late apoptosis and necrosis phases in comparison with their single concentrations, significantly. Metformin in combination with epirubicin and etoposide could arrest the S phase in B-CPAP and SW cells, significantly. Metformin in combination with epirubicin and etoposide could reduce ~100% migration rate, whereas single concentrations of epirubicin and etoposide could reduce ~50% migration rate. Combined treatment of metformin with anticancer drugs epirubicin and etoposide can increase the mortality in thyroid cancer cell lines and reduce the toxicity of these drugs on the normal cell line, which could be the starting point for proposing a new combination strategy in the therapy of thyroid cancer to induce more potency and reduce acute toxicity.

SiRNA targeting ABCB1 potentiates the efficacy of chemotherapy in human triple-negative breast cancer cells

Diminishing the efficacy of chemotherapy because of multidrug resistance (MDR) is a major clinical problem for triple-negative breast cancer (TNBC). MDR often occurs by overexpression of ATP-binding cassette B1 (ABCB1) protein that effuses various anticancer drugs from cancer cells. One of the newly developed techniques to addressing MDR is to knockdown ABCB1 by RNA interference (RNAi). RNAi is a gene-silencing process in that small interfering RNA (siRNA) blocks the expression of desired genes with high efficiency/specificity. The aim of this work is to examine the impact of ABCB1 inhibition via specific siRNAs on the efficacy of paclitaxel or etoposide in TNBC cells. The toxicity of increasing paclitaxel and etoposide concentrations on MDA-MB-231 cells was assessed using the MTT test. Cells were then co-treated with paclitaxel or etoposide in combination with ABCB1-siRNA, followed by cytotoxicity, colony formation, and migration assays. The administration of ABCB1-siRNA with paclitaxel or etoposide exhibited a synergistic effect and siRNA-drug treatments markedly reduced viability, clonogenicity, and migration of TNBC cells compared to siRNA or drug alone. Overall, these results indicate that TNBC cells become vulnerable even to sub-toxic doses of paclitaxel and etoposide after ABCB1-siRNA transfection, representing a promising aproach to enhance the influence of chemotherapy in TNBC.

Abstract 4204: CDK4/6 inhibition mitigates chemotherapy-induced immunosuppression and enhances the efficacy of chemo- immunotherapy in small cell lung cancer models

Abstract Small cell lung cancer (SCLC) is an aggressive form of lung cancer with a notably low survival rate. Chemotherapy and more recently its combination with immunotherapy are the standard of care for extensive stage SCLC. Although this combinatorial treatment provides clinical benefit, it can be hampered by the effect of chemotherapy on inducing immunosuppression. Cyclin dependent kinases 4/6 (CDK4/6) phosphorylate retinoblastoma (Rb) protein and promote proliferation of Rb-intact hematopoietic stem progenitor cells (HSPCs) by allowing cells to progress through S phase. Rb loss is a signature mutation in SCLC tumors, making these tumor cells unresponsive to CDK4/6 inhibition. Therefore, we hypothesized that CDK4/6 inhibitors could be utilized to block the proliferation of Rb-intact HSPCs and protected them from chemotherapy without altering the effect of chemotherapy on Rb-deficient SCLC cells. We treated Rb-deficient SCLC cancer cells and Rb-intact NK and T cells with increasing concentrations of cisplatin, etoposide and topotecan with or without the CDK4/6 inhibitor G1T28. Cell proliferation, apoptosis and cell cycle were analyzed using CellTiter-Glo, Annexin V staining, and PI-based cell cycle analysis. In vitro, CDK4/6 inhibition did not alter the impact of chemotherapy on Rb-deficient SCLC cell viability, whereas with Rb-intact NK and T cells, CDK4/6 inhibitor treatment induced cell cycle arrest and prevented apoptosis induced by chemotherapeutic agents. In the TKOTmG Rb-deficient mouse model of SCLC, CDK4/6 inhibition improved the efficacy of cisplatin plus etoposide. In vivo studies using the KP-1 syngeneic mouse models showed that treatment with a CDK4/6 inhibitor improved the efficacy of chemotherapy and immunotherapy individually or chemo-immunotherapy combinations by reducing the tumor growth rate and prolonging animal survival. CDK4/6i treatment prior to chemotherapy preserved white blood cell counts in comparison to chemotherapy alone. Tissue and blood immune marker analysis showed that CDK4/6 inhibitor treatment increased both the circulating and tumor-resident NK cells. Collectively, we demonstrated that CDK4/6 inhibition does not antagonize the therapeutic impact of chemotherapy on Rb-deficient SCLC cells but enhances the efficacy of chemotherapy and immunotherapy in KP-1 syngeneic SCLC mouse model. CDK4/6i also at least partially abrogated the chemotherapy-induced reductions in subpopulatons of immune cells. CDK4/6 inhibition may be considered as a therapeutic strategy for mitigating chemotherapy-induced immunosuppression and enhance antitumor immunity in SCLC patients. Citation Format: Thiruvengadam Arumugam, Irene G. Muñoz, Alissa Poteete, Fahoa Zhang, Monique Nilsson, John S. Yi, Jessica A. Sorrentino, Andrew P. Beelen, John V. Heymach. CDK4/6 inhibition mitigates chemotherapy-induced immunosuppression and enhances the efficacy of chemo- immunotherapy in small cell lung cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4204.

Abstract 1008: Cell density-related variability in chemotherapeutic resistance patterns in human cancer cells

Abstract Assessment of chemosensitivity and resistance varies with cancer cell of origin, drug and culture conditions. However, it is also not uncommon to find multiple different IC50 values for the same chemotherapeutic treatments acting on the same tumor cell lines. The aim of this research was to investigate this discrepancy in phenotypes and elucidate relevant variables that influence them. We assessed chemosensivity as a function of tumor cell density by plating TOV-21G ovarian, HT29 colorectal, and U2OS osteosarcoma cell lines in 96-well plates at various seeding densities per well and treated them with various concentrations of cisplatin, 5-fluorouracil (5-FU), and etoposide, respectively. CellTiter Glo assays were performed at 72 hours of treatment to determine IC50. Cisplatin IC50 for TOV-21G cells plated at 50,000, 20,000, 10,000, 5,000, 2,000, 1,000, and 500 cells/well were 12.00 μM, 5.47 μM, 3.16 μM, 2.45 μM, 2.54 μM, 2.83 μM, and 2.66 μM, respectively. Etoposide IC50 for U2OS cells plated at the same seeding densities were 6.50 μM, 5.19 μM, 3.23 μM, 2.98 μM, 2.49 μM, 2.13 μM, and 1.88 μM, respectively. IC50 values for HT29 cells treated with 5-FU at 2,000, 1,000, and 500 cells/well were 35.89 μM, 19.74 μM, and 13.42 μM, respectively. TOV-21G, U2OS, and HT29 cells all showed differences in IC50 values depending on cell plating density. TOV-21G and U2OS cells both showed a gradual decrease and plateau in IC50 as the cancer cell density decreased from 50,000 cells/well to 500 cells/well. HT29 cells displayed a sharper IC50 decrease as the cell density decreased from 2,000 cells/well to 500 cells/well. Our experiments demonstrate that IC50 values are not static at 72 hours and that tumor cell density is an important variable in influencing observed tumor resistance to traditionally effective drugs. As the density of cancer cells increases, the cells develop more resistance to the associated chemotherapy and require higher quantities of drug to achieve the same level of growth inhibition. We are currently exploring underlying molecular mechanisms that may influence these changes in IC50 including cell survival signaling proteins, cell-cell contact or growth factors that may influence cell density-related chemoresistance. Citation Format: Thomas Brown, Ujwal Punyamurtula, Jill Strandberg, Wafik S. El-Deiry. Cell density-related variability in chemotherapeutic resistance patterns in human cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1008.

The lack of functional DNMT2/TRDMT1 gene modulates cancer cell responses during drug-induced senescence.

Cellular senescence may be a side effect of chemotherapy and other anti-cancer treatments that may promote inflammation and paracrine secondary senescence in healthy tissues. DNMT2/TRDMT1 methyltransferase is implicated in the regulation of cellular lifespan and DNA damage response (DDR). In the present study, the responses to senescence inducing concentrations of doxorubicin and etoposide in different cancer cells with DNMT2/TRDMT1 gene knockout were evaluated, namely changes in the cell cycle, apoptosis, autophagy, interleukin levels, genetic stability and DDR, and 5-mC and NSUN1-6 levels. Moreover, the effect of azacytidine post-treatment was considered. Diverse responses were revealed that was based on type of cancer cells (breast and cervical cancer, osteosarcoma and glioblastoma cells) and anti-cancer drugs. DNMT2/TRDMT1 gene knockout in drug-treated glioblastoma cells resulted in decreased number of apoptotic and senescent cells, IL-8 levels and autophagy, and increased number of necrotic cells, DNA damage and affected DDR compared to drug-treated glioblastoma cells with unmodified levels of DNMT2/TRDMT1. We suggest that DNMT2/TRDMT1 gene knockout in selected experimental settings may potentiate some adverse effects associated with chemotherapy-induced senescence.

HGG-40. FOCUSED ULTRASOUND ENHANCES ETOPOSIDE DELIVERY IN A MURINE PONTINE GLIOMA MODEL

BackgroundDiffuse intrinsic pontine glioma (DIPG) is a devastating pediatric brain cancer with limited treatment options and poor survival. The delivery of systemic therapies in this disease is severely limited by the blood-brain barrier (BBB). Focused ultrasound combined with intravenous microbubbles (FUS+MB) can effectively open the BBB permitting the entry of drugs across the cerebrovasculature. Etoposide is a chemotherapy frequently used in pediatric oncology with well-established anti-tumor effects but limited efficacy when administered systemically in DIPG. Given that FUS+MB in DIPG is not well studied, our goal was to determine the feasibility of ultrasound-mediated BBB opening and etoposide delivery in a preclinical murine pontine glioma model. MethodsA syngeneic, orthotopic model was established by stereotactic injection of PDGF-B+PTEN-/-p53-/- murine glioma cells into the pons of B6 albino mice. Mice were randomly divided into control (n=6) or FUS+MB groups (n=6). A single-element, spherical-segment FUS transducer (center frequency=1.5MHz) driven by a function generator through a power amplifier was used with concurrent microbubble injection to sonicate the tumor and its margins on post-injection day 14. Immediately after treatment, 5 mg/kg of intraperitoneal etoposide was administered to all 12 mice. All animals underwent cardiac puncture and blood sampling, followed by transcardiac perfusion and brain harvesting. Liquid chromatography-mass spectometry was performed on both serum and tumor tissue to measure etoposide levels. ResultsContrast-enhanced MRI demonstrated successful BBB opening in all FUS+MB mice. Compared to control (mean=20.98ng/g), etoposide concentration in the sonicated tumor tissue (mean=164.77ng/g) was nearly eight times greater. Lastly, the mean brain tumor–to-serum ratio was more than fivefold higher in the treated mice (1.50%) compared with the control mice (0.28%) (P<0.005). ConclusionsFUS+MB is hereby shown successful in BBB opening and enhanced delivery of etoposide in a preclinical pontine glioma model.

EIF4F Inhibition Sensitizes Non‐Small Cell Lung Cancer Cells to a Topoisomerase II Inhibitor

Lung cancer is one of the most common and deadly cancers worldwide. In the past, Etoposide (ETP), a topoisomerase II inhibitor, has been used to treat non-small cell lung cancer (NSCLC). However, high concentrations of ETP have been shown to have considerable adverse effects. Combining ETP with another drug as a chemosensitizer could increase the efficacy of ETP. Combination therapies with ETP have been largely unexplored. In past studies, NSCLC has been treated with a novel drug compound, 4EGI-1, a translation inhibitor. By chemosensitizing NSCLC with 4EGI-1, we hypothesized that we could decrease the amount of ETP needed for effective treatment. Here we examine the relationship between different concentrations of ETP and 4EGI-1 and their effect on lung cancer cells. Cell death was used as an assay to determine the efficacy of the agents separately and in combination. The combined effects of ETP and 4EGI-1 on NSCLC were observed to reduce cell viability by 76-84%, a significant increase from using no treatment, or using either drug alone. ETP alone reduced cell viability by 52%, and 4EGI-1 alone reduced cell viability by 52-64%. From the data, we can conclude that combining ETP and 4EGI-1 compared to using either drug alone led to significantly lower cell viability (Student's T-test, p < 0.02). This in vitro study demonstrates the improved activity of ETP with the addition of the translation inhibitor 4EGI-1.

Inhibition of Aedes aegypti DNA topoisomerase II by etoposide: Impact on survival and morphology of larvae and pupae

DNA topoisomerase II enzymes maintain DNA stability during vital processes, such as genome replication, transcription and chromosomal segregation during mitosis and meiosis. In the present work, we analyzed functional aspects of the DNA topoisomerase II (AeTopII) enzyme of the mosquito Aedes aegypti. Here, we show that AeTopII mRNA is expressed at all stages of mosquito development. By in situ hybridization, we found that the AeTopII mRNA is concentrated along the ovarian follicular cells as well as in the region of the follicles. The observed expression profiles likely reflect increased topoisomerase II cellular requirements due to the intense ovarian growth and egg production following blood feeding in Ae. aegypti females. The drug etoposide, a classic inhibitor of topoisomerase II, was used for in vivo testing with 2nd stage larvae, in order to investigate the functional importance of this enzyme in Ae. aegypti survival and development. Inhibition of topoisomerase II activity with etoposide concentrations ranging from 10 to 200 μM did not leads to the immediate death of larvae. However, after 10 days of observation, etoposide treatments resulted in 30–40% decrease in survival, in a dose dependent manner, with persisting larvae and pupae presenting incomplete development, as well as morphological abnormalities. Also, approximately 50% of the treated larvae did not reach the pupal stage. Thus, we conclude that AeTopII is a vital enzyme in the development of Ae. aegypti and its sensitivity to inhibitors should be explored for potential chemical agents to be used in vector control.

Focused ultrasound mediated blood\u2013brain barrier opening is safe and feasible in a murine pontine glioma model

Drug delivery in diffuse intrinsic pontine glioma is significantly limited by the blood-brain barrier (BBB). Focused ultrasound (FUS), when combined with the administration of microbubbles can effectively open the BBB permitting the entry of drugs across the cerebrovasculature into the brainstem. Given that the utility of FUS in brainstem malignancies remains unknown, the purpose of our study was to determine the safety and feasibility of this technique in a murine pontine glioma model. A syngeneic orthotopic model was developed by stereotactic injection of PDGF-B+PTEN−/−p53−/− murine glioma cells into the pons of B6 mice. A single-element, spherical-segment 1.5 MHz ultrasound transducer driven by a function generator through a power amplifier was used with concurrent intravenous microbubble injection for tumor sonication. Mice were randomly assigned to control, FUS and double-FUS groups. Pulse and respiratory rates were continuously monitored during treatment. BBB opening was confirmed with gadolinium-enhanced MRI and Evans blue. Kondziela inverted screen testing and sequential weight lifting measured motor function before and after sonication. A subset of animals were treated with etoposide following ultrasound. Mice were either sacrificed for tissue analysis or serially monitored for survival with daily weights. FUS successfully caused BBB opening while preserving normal cardiorespiratory and motor function. Furthermore, the degree of intra-tumoral hemorrhage and inflammation on H&E in control and treated mice was similar. There was also no difference in weight loss and survival between the groups (p > 0.05). Lastly, FUS increased intra-tumoral etoposide concentration by more than fivefold. FUS is a safe and feasible technique for repeated BBB opening and etoposide delivery in a preclinical pontine glioma model.

Focused Ultrasound-Mediated Blood-Brain Barrier Opening Increases Delivery and Efficacy of Etoposide for Glioblastoma Treatment

Glioblastoma (GBM) is a devastating disease. With the current treatment of surgery followed by chemoradiation, outcomes remain poor, with median survival of only 15 months and a 5-year survival rate of 6.8%. A challenge in treating GBM is the heterogeneous integrity of the blood-brain barrier (BBB), which limits the bioavailability of systemic therapies to the brain. There is a growing interest in enhancing drug delivery by opening the BBB with the use of focused ultrasound (FUS). We hypothesize that an FUS-mediated BBB opening can enhance the delivery of etoposide for a therapeutic benefit in GBM. A murine glioma cell line (Pdgf+, Pten-/-, P53-/-) was orthotopically injected into B6(Cg)-Tyrc-2J/J mice to establish the syngeneic GBM model for this study. Animals were treated with FUS and microbubbles to open the BBB to enhance the delivery of systemic etoposide. Magnetic resonance (MR) imaging was used to evaluate the BBB opening and tumor progression. Liquid chromatography tandem mass spectrometry was used to measure etoposide concentrations in the intracranial tumors. The murine glioma cell line is sensitive to etoposide invitro. MR imaging and passive cavitation detection demonstrate the safe and successful BBB opening with FUS. The combined treatment of an FUS-mediated BBB opening and etoposide decreased tumor growth by 45% and prolonged median overall survival by 6 days: an approximately 30% increase. The FUS-mediated BBB opening increased the brain tumor-to-serum ratio of etoposide by 3.5-fold and increased the etoposide concentration in brain tumor tissue by 8-fold compared with treatment without ultrasound. The current study demonstrates that BBB opening with FUS increases intratumoral delivery of etoposide in the brain, resulting in local control and overall survival benefits.

In Vitro Cytotoxicity Study of Cyclophosphamide, Etoposide and Paclitaxel on Monocyte Macrophage Cell Line Raw 264.7.

The presence of antineoplastic compounds in aquatic ecosystem is an emerging challenge for the society. Antineoplastic compounds released into the aquatic environment exhibit a potential threat to normal aquatic life. Particularly, antineoplastic compounds are responsible for direct or indirect interference with the cellular DNA of an organism and cause toxicity to cells. The present study focused on the assessment of in vitro toxic effect of cyclophosphamide, etoposide and paclitaxel on Raw 264.7 cell line (mouse monocyte macrophage cells). The inhibitory concentration of cyclophosphamide, etoposide, and paclitaxel was determined. The IC50 values of these compounds were 145.44, 5.40, and 69.76µgml-1 respectively. This is the first report on toxicity analysis of cyclophosphamide, paclitaxel and etoposide on Raw 264.7 cell line by reducing cell viability and indicating the cell cytotoxicity i.e., 69.58% for cyclophosphamide, 92.01% for etoposide and 88.85% for paclitaxel on concentration 250µgml-1. The results of their cytotoxicity assessment highlight the need of improvement in sewage treatment technology for the efficient removal of these compounds from aquatic environment.

Target specific intracellular quantification of etoposide by quadrupole-time of flight based mass spectrometric method

Etoposide (ETP), a widely used chemotherapeutic agent has an intracellular target site of action. Unfortunately, the concentration of ETP in plasma does not properly reflect the concentration in its intracellular site of action. As per our knowledge, no reported bioanalytical method is available for intracellular quantification of ETP. In this research, we developed an LC-MS/MS method to quantitate ETP in intracellular compartments of MCF-7 cells. The Abcam nuclear extraction kit was used for extracting the nuclear and cytosolic protein from MCF-7 cells. The method showed excellent linearity in the 20–1000 ng/mL range. The intra and inter-day precision (%CV) including LLOQ were found to be in the range of 2.19–16.96% and 6.71–11.21%, respectively, with an accuracy of 86.87 to 110.37% and 93.03 to 100.50%, respectively. The concentration of ETP in nuclear and cytosolic fraction was successfully quantitated using the developed method. The developed method can be applied to understand the efficacy of different formulations based on the intracellular ETP concentration in vitro. It can be considered as a model method for quantification of other similar categories of drugs in their actual intracellular site of action after required optimization in the methodology.