Cellular Viability Assays Research Articles

A potential role for hepcidin in obesity-driven colorectal tumourigenesis.

The obesity epidemic is associated with increases in the incidence of several types of cancer, including colorectal cancer, and is associated with poor outcomes for patients. Adipose tissue is considered biologically active and represents a plausible link between cancer and obesity due to the many factors that it secretes. In the present study, human adipose tissue was cultured invitro and predifferentiated adipocyte secretome[preadipocyte (PAS)] and differentiated adipocyte secretome(DAS) were collected. Quantification of interleukin-6(IL-6), leptin and hepcidin in the DAS medium was compared to the PAS medium. Fold change levels of hepcidin, leptin and IL-6 in DAS (2.88±0.28, 12.34±0.95 and 31.29±1.89 fold increases) were significantly higher compared to these in PAS (p=0.05). The SW480 colorectal cancer cells were co-cultured with DAS in the presence or absence of leptin, IL-6 or hepcidin inhibitors and cellular viability and proliferation assays were performed. The culture of SW480 with DAS increased the cell proliferation and viability by 30and15% (p=0.02 and p=0.03) respectively, which was reversed in the presence of inhibitors. Challenging the SW480 cells with IL-6 or hepcidin significantly elevated colonocyte‑secreted leptin(p=0.05). Challenging the SW480 cells with leptin or hepcidin resulted in elevated levels of colonocyte-secreted IL-6(p=0.05). Similarly, challenging cells with either IL-6 or leptin markedly elevated the level of secreted hepcidin(p=0.05) and this was associated with an induction in colonocyte iron levels in both cases. Collectively, these data revealed that adipocyte-secreted factors can ultimately modulate colonocyte iron levels and phenotype.

Abstract 221: Integrated functional proteomics of MET/VEGFR inhibitors reveals complex mechanism of action of foretinib in NSCLC

Abstract BACKGROUND: Foretinib (FORE) and cabozantinib (CABO) are two MET/VEGFR inhibitors with similar chemical structures. CABO is FDA-approved for medullary thyroid and renal cancer; in addition, it is in clinical trials for treatment of non-small cell lung cancer (NSCLC). Through an unbiased viability screen we have observed potent cellular activity of FORE, but not CABO, in several NSCLC cell lines. We have previously shown that most NSCLC cell lines are insensitive to MET or VEGFR inhibition, suggesting off-target activity of FORE in these cells. The aim of this project is to identify the mechanism of action of FORE in NSCLC and design an optimized combination therapy. METHODS: Cellular viability assays were done using CellTiter-Glo, cell cycle analysis by flow cytometry. Western blotting was performed to evaluate the induction of apoptosis through PARP1 and caspase cleavage, as well changes in signaling. We synthesized FORE and CABO analogues and performed differential quantitative chemical and phosphoproteomics to determine the target kinase profile and pathway effects in NSCLC cells. Changes in gene expression upon drug treatment were measured by RNA-seq. RNAi in combination with pharmacological inhibitors was performed to interrogate targets and pathways. RESULTS: FORE showed greater potency in NSCLC cell lines than CABO with regard to inhibition of viability and induction of apoptosis. FORE decreased phosphorylation of AKT and ERK. Chemical and phosphoproteomics revealed several kinases, such as MEK and MAP4K5, to bind preferentially by FORE over CABO that differentially affect the adherens junction and MAPK signaling pathways. Target validation showed differential inhibition of MEK1/2 and MAP4K5. Cellular validation with RNAi in combination with pharmacological inhibitors suggested that MEK1/2, MAP4K5 and IGF1R are involved in the mechanism of action of foretinib in NSCLC cells. RNA-seq pathway analysis furthermore suggested regulation of chromatin organization and Wnt pathway signaling by foretinib. CONCLUSION: Our results suggest that the difference in the efficacy between FORE and CABO is related to polypharmacology of FORE, which simultaneously targets IGF1R, MEK1/2 and MAP4K5. This difference results in divergence in signaling pathway inhibition and induces distinct effects in NSCLC. The establishment of FORE targets and signaling pathways can lead to optimized combination therapy for NSCLC and identification of new actionable kinases in lung cancer cells. Citation Format: Natalia J. Sumi, Bin Fang, Lily L. Rix, Muhammad Ayaz, Fumi Kinose, Eric A. Welsh, Steven A. Eschrich, Harshani R. Lawrence, John M. Koomen, Eric B. Haura, Uwe Rix. Integrated functional proteomics of MET/VEGFR inhibitors reveals complex mechanism of action of foretinib in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 221. doi:10.1158/1538-7445.AM2017-221

Abstract 3233: Design and synthesis of BPR1K653 and its derivatives targeting the back pocket of Aurora kinases for selective isoform inhibition

Abstract Guided by computational modeling addressing the back pocket of Aurora kinases, several novel chemical analogs of BPR1K653 were designed and synthesized in two steps with 30-66% yields. All synthesized compounds were evaluated by Aurora-A and Aurora-B enzymatic kinase activity assays, cellular phosphorylation inhibition assays and cell viability assays. Of them, compound 3a that exhibits no substitution at phenylurea showed similar inhibition activity to Aurora-A and -B. Compounds 3b and 3c which harbor tertiary amino group at meta position of phenylurea showed 10-15 fold inhibition selectivity for Aurora-A over Aurora-B in enzymatic assays. In addition, 3b and 3c were more than 200 fold superior in inhibiting T-loop autophosphorylation of Aurora-A compared to Aurora-B in HCT116 colon carcinoma cells. On the other hand, compound 3d with a 4-hydroxypiperidinyl group at ortho position preserves Aurora-B inhibition activity. Molecular docking study revealed that the various steric interaction between the compound and residues in the back pocket of Aurora kinases determines their inhibition selectivity. Citation Format: Ya-Hui Chi, Chun-Ping Chang, Yi-Yu Ke, Wen-Hsing Lin, Hsing-Pang Hsieh. Design and synthesis of BPR1K653 and its derivatives targeting the back pocket of Aurora kinases for selective isoform inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3233. doi:10.1158/1538-7445.AM2017-3233

Abstract 4025: Accelerating drug repurposing for cancer therapy using multiplexed viability assays

Abstract New therapies are desperately needed for patients with advanced cancer, but making safe and efficacious drugs remains expensive and time consuming. Meanwhile, multiple non-oncology drugs have been successfully repurposed for cancer, including thalidomide for multiple myeloma and aspirin for prevention of colorectal cancer. While the benefits of repurposing are clear, successes to date have been largely serendipitous. We have created two foundational tools to identify drug repurposing opportunities at a much greater scale: a new world-class screening library of more than 4,000 drugs/clinical candidates and a multiplexed method to rapidly conduct cellular viability assays across hundreds of cell lines. PRISM, a recently developed high-throughput assay, employs DNA-barcoded cell lines to enable rapid testing of many drugs against pools of cancer cell lines. We have tested 4,100 compounds against 578 genomically characterized cancer cell lines using PRISM. Our experiment is among the largest cell line screens ever undertaken. Global analysis has revealed multiple strong clusters of active drugs, including vitamin D agonists, bromodomain inhibitors, and statins. In addition, many novel and established drug-biomarker relationships were identified, including alkylating agent response and low MGMT expression, BRAF inhibitor response and BRAF mutation, and MDM2 inhibitor response and TP53 wild-type status. Surprisingly, more than 100 non-oncology drugs unexpectedly killed multiple cancer cell lines, making them attractive repurposing candidates. For example, the FDA-approved phosphodiesterase inhibitor anagrelide selectively kills PDE3A-high lung cancer and melanoma cell lines. Confirmatory experiments are underway to validate screening results, evaluate putative biomarkers, and test drug activity in preclinical models. Importantly, this approach is expected to enable rapid initiation of clinical trials, dramatically accelerating patient access to potential new therapies. Citation Format: Steven M. Corsello, Christopher C. Mader, Jordan Bryan, Jennifer Roth, David Peck, John Davis, Samantha Bender, Li Wang, Alice Wang, Joshua Bittker, Francisca Vazquez, Aravind Subramanian, Aviad Tsherniak, Todd R. Golub. Accelerating drug repurposing for cancer therapy using multiplexed viability assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4025. doi:10.1158/1538-7445.AM2017-4025

Molecular cloning, characterization and expression analysis of Frizzled 6 in the small intestine of pigs (Sus scrofa).

Frizzled 6 (FZD6) encodes an integral membrane protein that functions in multiple signal transduction pathways, for example, as a receptor in Wnt/planar cell polarity (PCP) signaling pathway for polarized cell migration and organ morphogenesis. Mutations in FZD6 have been identified in a variety of tumors. In this study, the full-length cDNA of Sus scrofa FZD6 (Sfz6) was cloned and characterized. Nucleotide sequence analysis demonstrates that the Sfz6 gene encodes the 712 amino-acid (aa) protein with seven transmembrane domain. Tissue distribution analysis showed that Sfz6 mRNA is ubiquitously expressed in various tissues, being highest in kidney, moderate in jejunum, ileum, colon, liver, and spleen. However, FZD6 protein is highly expressed in the heart and there was no significant difference in other tissues. The relative abundance and localization of FZD6 protein in jejunum along the crypt-villus axis was determined by Western blot and immunohistochemical localization. The results show that in the jejunum, FZD6 protein is highly expressed in the villus and less in the crypt cells. Cellular proliferation and viability assays indicate that knockdown of FZD6 with small interfering RNAs (siRNA) significantly reduced the cell viability of the intestinal porcine enterocyte cells (IPEC-J2). Furthermore, qPCR and Western blot analysis revealed that expressions of ras-related C3 botulinum toxin substrate 1 (Rac1); ras homolog gene family member A (RhoA) and c-Jun N-terminal kinase 1 (JNK1), some components of PCP signaling pathway were upregulated (P < 0.05) by knockdown of FZD6 in IPEC-J2 cells. In conclusion, these results showed that FZD6 abundance in the villus was higher than that in crypt cells and knockdown of FZD6 induces PCP signal pathway components expression in IPEC-J2 cells. Our findings provide the foundation for further investigation into porcine FZD6 gene.

Poly(N-(4-aminobutyl)-acrylamide) as mimetic polylysine for improving survival and differentiation of cerebellar granule neurons.

Poly(N-(4-aminobutyl)-acrylamide) (P4Am) and poly-d-lysine (PDL) possess the same butylamine side chain. The main difference is the peptide structure, which is in the main chain of PDL but in the side chain of P4Am. PDL has been extensively used in the preparation of neuronal cultures. We assumed neurons are sensitive enough to distinguish such structure difference so these two cationic polymers were compared at serial coating concentrations for culturing cerebellar granule neurons from 7-day-old Wistar rats in this study. Cellular viability and morphology assay showed no obvious difference for neurons cultured at high coating concentrations (>0.31 µg/mL) of these two polymers. In contrast, the difference in the peptide structure between P4Am and PDL could be distinguished by neurons at low coating concentrations (< 0.16 µg/mL). P4Am at low coating concentration could keep aggregates with three or four thick processes to support a more complete neural network with higher cellular viability than PDL. This suggests the interaction between neurons and the specific peptide structure of P4Am at low coating concentration was able to improve survival and differentiation of cultured cerebellar granule neurons. Although neural cells exhibited different morphologies and activities on high and low P4Am coating concentrations, immunofluorescence imaging confirmed most of cultured cells were functionally mature neurons, stained by neurofilament, synapsin I, and GAP43. The information should be useful for designing new biomaterials for regeneration of damaged circuits following disease or trauma. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1194-1201, 2018.

Lipoic Acid Gold Nanoparticles Functionalized with Organic Compounds as Bioactive Materials.

Water soluble gold nanoparticles protected by lipoic acid were obtained and further functionalized by standard coupling reaction with 1-naphtylamine, 4-aminoantipyrine, and 4′-aminobenzo-15-crown-5 ether. Derivatives of lipoic acid with 1-naphtylamine, 4-aminoantipyrine, and 4′-aminobenzo-15-crown-5 ether were also obtained and characterized. All these were tested for their antimicrobial activity, as well as for their influence on mammalian cell viability and cellular cycle. In all cases a decreased antimicrobial activity of the obtained bioactive nanoparticles was observed as compared with the organic compounds, proving that a possible inactivation of the bioactive groups could occur during functionalization. However, both the gold nanoparticles as well as the functionalized bioactive nanosystems proved to be biocompatible at concentrations lower than 50 µg/mL, as revealed by the cellular viability and cell cycle assay, demonstrating their potential for the development of novel antimicrobial agents.

Modified SEM Preparation for THP-1 Monocytes

THP-1 monocytes are non-adherent white blood cells that are typically differentiated into macrophage like cells to induce cellular adherence for cellular viability assays. However, the induced differentiation also alters the external cellular morphology of THP-1 cells, therefore protocol modification and development has led to a novel method of preparation of THP-1 monocytes for scanning electron microscopy. Incorporation of a polystyrene substrate, modification of the dehydration gradient and removal of critical point drying has been shown effective in preparing THP-1 monocytes for observation without alteration of external structure. Analysis of cellular integrity was subsequently via treatment with 100 nm recombinant Panton-Valentine leukocidin to induce cell lysis and allow observation of internal cellular composition and quality of preservation of the proposed preparatory protocol.

Identification of therapeutic targets applicable to clinical strategies in ovarian cancer.

BackgroundshRNA-mediated lethality screening is a useful tool to identify essential targets in cancer biology. Ovarian cancer (OC) is extremely heterogeneous and most cases are advanced stages at diagnosis. OC has a high response rate initially, but becomes resistant to standard chemotherapy. We previously employed high throughput global shRNA sensitization screens to identify NF-kB related pathways. Here, we re-analyzed our previous shRNA screens in an unbiased manner to identify clinically applicable molecular targets.MethodsWe proceeded with siRNA lethality screening using the top 55 genes in an expanded set of 6 OC cell lines. We investigated clinical relevance of candidate targets in The Cancer Genome Atlas OC dataset. To move these findings towards the clinic, we chose four pharmacological inhibitors to recapitulate the top siRNA effects: Oxozeaenol (for MAP3K7/TAK1), BI6727 (PLK1), MK1775 (WEE1), and Lapatinib (ERBB2). Cytotoxic effects were measured by cellular viability assay, as single agents and in 2-way combinations. Co-treatments were evaluated in either sequential or simultaneous exposure to drug for short term and extended periods to simulate different treatment strategies.ResultsLoss-of-function shRNA screens followed by short-term siRNA validation screens identified therapeutic targets in OC cells. Candidate genes were dysregulated in a subset of TCGA OCs although the alterations of these genes showed no statistical significance to overall survival. Pharmacological inhibitors such as Oxozeaenol, BI6727, and MK1775 showed cytotoxic effects in OC cells regardless of cisplatin responsiveness, while all OC cells tested were cytostatic to Lapatinib. Co-treatment with BI6727 and MK1775 at sub-lethal concentrations was equally potent to BI6727 alone at lethal concentrations without cellular re-growth after the drugs were washed off, suggesting the co-inhibition at reduced dosages may be more efficacious than maximal single-agent cytotoxic concentrations.ConclusionsLoss-of-function screen followed by in vitro target validation using chemical inhibitors identified clinically relevant targets. This approach has the potential to systematically refine therapeutic strategies in OC. These molecular target-driven strategies may provide additional therapeutic options for women whose tumors have become refractory to standard chemotherapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2675-5) contains supplementary material, which is available to authorized users.

Gold Nanoantenna-Mediated Photothermal Drug Delivery from Thermosensitive Liposomes in Breast Cancer.

In this work, we demonstrate controlled drug delivery from low-temperature-sensitive liposomes (LTSLs) mediated by photothermal heating from multibranched gold nanoantennas (MGNs) in triple-negative breast cancer (TNBC) cells in vitro. The unique geometry of MGNs enables the generation of mild hyperthermia (∼42 °C) by converting near-infrared light to heat and effectively delivering doxorubicin (DOX) from the LTSLs in breast cancer cells. We confirmed the cellular uptake of MGNs by using both fluorescence confocal Z-stack imaging and transmission electron microscopy (TEM) imaging. We performed a cellular viability assay and live/dead cell fluorescence imaging of the combined therapeutic effects of MGNs with DOX-loaded LTSLs (DOX-LTSLs) and compared them with free DOX and DOX-loaded non-temperature-sensitive liposomes (DOX-NTSLs). Imaging of fluorescent live/dead cell indicators and MTT assay outcomes both demonstrated significant decreases in cellular viability when cells were treated with the combination therapy. Because of the high phase-transition temperature of NTSLs, no drug delivery was observed from the DOX-NTSLs. Notably, even at a low DOX concentration of 0.5 μg/mL, the combination treatment resulted in a higher (33%) cell death relative to free DOX (17% cell death). The results of our work demonstrate that the synergistic therapeutic effect of photothermal hyperthermia of MGNs with drug delivery from the LTSLs can successfully eradicate aggressive breast cancer cells with higher efficacy than free DOX by providing a controlled light-activated approach and minimizing off-target toxicity.

SYD985, a Novel Duocarmycin-Based HER2-Targeting Antibody-Drug Conjugate, Shows Antitumor Activity in Uterine Serous Carcinoma with HER2/Neu Expression.

Uterine serous carcinoma (USC) is an aggressive form of endometrial cancer. Up to 35% of USC may overexpress the HER2/neu oncogene at strong (i.e., 3+) levels by IHC while an additional 40% to 50% express HER2/neu at moderate (2+) or low (1+) levels. We investigated the efficacy of SYD985, (Synthon Biopharmaceuticals), a novel HER2-targeting antibody-drug conjugate (ADC) composed of the mAb trastuzumab linked to a highly potent DNA-alkylating agent (i.e., duocarmycin) in USC. We also compared the antitumor activity of SYD985 in head-to-head experiments to trastuzumab emtansine (T-DM1), a FDA-approved ADC, against multiple primary USC cell lines expressing different levels of HER2/neu in in vitro and in vivo experiments. Using antibody-dependent cellular cytotoxicity (ADCC), proliferation, viability, and bystander killing assays as well as propidium iodide-based flow cytometry assays and multiple in vivo USC mouse xenograft models, we demonstrate for the first time that SYD985 is a novel ADC with activity against USC with strong (3+) as well as low to moderate (i.e., 1+/2+) HER2/neu expression. SYD985 is 10- to 70-fold more potent than T-DM1 in comparative experiments and, unlike T-DM1, it is active against USC demonstrating moderate/low or heterogeneous HER2/neu expression. Clinical studies with SYD985 in patients harboring chemotherapy-resistant USC with low, moderate, and high HER2 expression are warranted. Mol Cancer Ther; 15(8); 1900-9. ©2016 AACR.

Abstract B46: miRNA-193a-5p repression of p73 induces Cisplatine chemoresistance in bone-related sarcomas

Abstract The purpose of this study is to better delineate the implication of the TAp73β/miRNA-193a-5p's axis in the Cisplatine chemoresistance, in the context of Osteosarcoma and Ewing Sarcoma, the two most common Bone Sarcomas. The last decade's improvement in the biomedical research field has widely contributed to improve the Bone Sarcomas outcome. Among the arsenal of chemotherapeutic-drugs currently used in those pathologies, the Cisplatine is a very common therapeutic option. Unfortunately, chemoresistance against this drug remains, often leading to the development of pulmonary metastases and the consequent relapse of the young patients suffering from these tumors. In the hope of eradicating these aggressive kinds of cancer, a better understanding of the molecular origins of such chemoresistance is needed. Through their gene's expression inhibitory role, the implication of the miRNAs in the Bone Sarcomas' chemoresistance is already clearly established. Furthermore, it has previously been demonstrated that through targeting the p53-family member TAp73β, the miR-193a-5p is implicated in the Cisplatine resistance in a carcinoma model. Thus, the aim of this work is to assess if TAp73β is also a mediator of the Cisplatine-induced apoptosis in Bone Sarcomas and consequently, if its modulation by the miR-193a-5p could be implicated in the chemoresistance mechanisms against this drug. In addition, this study also strives to bring the proof of concept of miR-193a-5p's as a new therapeutic-target to potentiate the Cisplatine anti-tumor's effects in Bone Sarcomas and override chemoresistance. Our qRT-PCR and viability assays results reveal that human Osteosarcoma and Ewing Sarcoma cell lines express the miR-193a-5p and TAp73β at various levels and display different Cisplatine sensitivities. In addition, an interesting correlation was found between the Cisplatine IC50 values and the miR-193a-5p expression levels. In line with these results, we have also demonstrated for the first time in this model that Cisplatine represses the miR-193a-5p expression and consequently induces TAp73β and its target-genes. Through TAp73β expression level modulation, we validated its implication in both the apoptosis and the clonogenic capabilities of the Bone Sarcoma cells. Our cellular viability assays also confirm the predicted role of TAp73β-mediated Cisplatine-sensitivity. In the same way, we show that modulating the miR-193a-5p expression level through transient transfections with synthesis miRNAs (pre-miR) or their inhibitory counterparts (anti-miRs) has a functional impact on the Cisplatine sensitivity and the apoptosis-induction (caspase 3/7 activity assay, PARPs cleavage). In conclusion, our body of data confirms that through targeting TAp73β, the miRNA-193a-5p modulates the viability, the clonogenic capabilities and the Cisplatine-induced apoptosis in Bone Sarcoma cells. Such results shed light on the implication of the miR-193a-5p in the Bone Sarcomas' Cisplatine-chemoresistance and open the road to new therapeutic opportunities provided by targeting the miR-193a-5p/TAp73β axis in the context of these pediatric cancers. In a fundamental approach, studying the Cisplatine-resistance might lead to better understand the role of miRNAs in therapeutic resistance. However, this miRNAs could also be used as “chemo-sensitizer” molecule in this context, improving the Bone Sarcoma outcome. Citation Format: Camille Jacques, François Lamoureux, Marc Baud'huin, Lidia Rodriguez Calleja, Dominique Heymann, Benjamin Ory. miRNA-193a-5p repression of p73 induces Cisplatine chemoresistance in bone-related sarcomas. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B46.

Multiparametric luminescent cell viability assay in toxicology models: A critical evaluation

IntroductionIn cellular viability assays the sole determination of a single parameter might not give precise information on the extent of toxicity. In our study we worked out a multiparametric microplate assay based on bioluminescent ATP quantification, esterase activity-related fluorescence, nucleic acid staining and total intracellular protein measurement from the same sample in MDCK and HepG2 tissue cultures. MethodsDose–response analyses were done after ATP depletion by metabolic poisons (NaF, NaN3) and by ochratoxin A (OTA) mycotoxin treatments. A novel perchloric acid fixation/extraction technique was applied in order to obtain intracellular ATP levels, esterase activity, DNA content and protein data simultaneously. Esterase activity was assessed by a fluorogenic staining. Estimation of cell number was done by DAPI fluorescence. Our results were expressed as ATP/protein, calcein fluorescence/ATP, calcein fluorescence/protein and ATP/DAPI ratios. Apoptosis/necrosis rates were measured by Annexin V–propidium iodide and 7-aminoactinomycin D flow cytometric assays and effects of OTA on actin cytoskeleton were also studied by using labeled phalloidin for visualization of actin. ResultsWe could verify that the esterase assay was not an energy driven (true viability) process. ATP/protein, calcein fluorescence/ATP, calcein fluorescence/protein ratios, DAPI fluorescence and protein levels together with morphological and apoptosis/necrosis parameters deciphered subtle changes in cell viability with good between-run precision. Dose dependent loss in cell number and decreased protein levels were observed in all cases, while disorganization of actin microfilaments was seen in OTA treated cells. The two cell lines did not respond uniformly to the same treatments. DiscussionATP/protein ratio proved to be a useful viability parameter however, the suppression and/or loss of intracellular protein could cause difficulty in interpreting ATP/protein data. We conclude that correct assessment of cellular viability should be done by measuring multiple parameters related to the specific mode of action of the tested toxic compound.

Flurbiprofen-loaded Nanoparticles Can Cross a Primary Porcine In vitro Blood-brain Barrier Model to Reduce Amyloid-β42 Burden

Elevated amyloid-β42 (Aβ42) in the brain is expected to cause Alzheimer’s Disease (AD). Reducing Aβ42 is therefore a cornerstone in causal drug development. Nevertheless, many promising substances failed in clinical trials, because reaching the target organ in vivo is difficult. The brain is protected by the Blood-Brain Barrier (BBB) that shields off most molecules to maintain the brain homeostasis. Brain-targeted nanoparticles are one successful tool to bypass this problem: by acting as Trojan horses they carry embedded drugs across the BBB for brain disorder treatment. Here, flurbiprofen, a γ-secretase modulator, was embedded in Poly(Lactic Acid) (PLA) nanoparticles. We tested if the drug-loaded nanoparticles affected the integrity of our advanced in vitro BBB model in transendothelial electrical resistance measurements and permeability assays, and investigated the nanoparticle-cell interaction in flow cytometry and confocal laser scanning microscopy. Furthermore, we assessed the drug transport capacity by highperformance liquid chromatography and the biological efficacy of the embedded drug in an Aβ42-detecing ELISA. We also verified the viability of the AD model cells by a cellular viability assay. After adding flurbiprofen-loaded nanoparticles to the blood compartment of a Transwell® model, the drug was detectable in the brain compartment, where it induced an Aβ42 lowering effect. Flurbiprofen from nanoparticles crossed the BBB without impairing barrier integrity, whereas the free drug was highly cytotoxic and destroyed the barrier. Ligand coupling of apolipoprotein E3 to the nanoparticles increased cellular uptake. Hence, we expect an even more pronounced Aβ42 reducing effect for apolipoprotein-modified, flurbiprofen-loaded nanoparticles. In conclusion, we enabled transport of a hardly permeable drug across an advanced in vitro BBB model, opening opportunities in the treatment and prevention of AD and other brain disorders. Using a primary porcine BBB model that displays excellent barrier characteristics, we show that flurbiprofen-loaded nanoparticles reduce Aβ42 burden without impairing barrier function.

All-trans retinoic acid protects against doxorubicin-induced cardiotoxicity by activating the ERK2 signalling pathway.

Doxorubicin is a powerful antineoplastic agent for treating a wide range of cancers. However, doxorubicin cardiotoxicity of the heart has largely limited its clinical use. All-trans retinoic acid (ATRA) plays an important role in many cardiac biological processes, but its protective effects on doxorubicin-induced cardiotoxicity remain unknown. Here, we studied the effect of ATRA on doxorubicin cardiotoxicity and the underlying mechanisms. Cellular viability assays, Western blotting and mitochondrial respiration analyses were employed to evaluate the cellular response to ATRA in H9c2 cells and primary cardiomyocytes. Quantitative PCR and gene knockdown were performed to investigate the underlying molecular mechanisms of ATRA's effects on doxorubicin cardiotoxicity. ATRA significantly inhibited doxorubicin-induced apoptosis in H9c2 cells and primary cardiomyocytes. ATRA was more effective against doxorubicin cardiotoxicity than resveratrol and dexrazoxane. ATRA also suppressed reactive oxygen species generation and restored expression levels of mRNA and proteins in the phase II detoxifying enzyme system: nuclear factor-E2-related factor 2, manganese superoxide dismutase, haem oxygenase-1, and mitochondrial function (mitochondrial membrane integrity, mitochondrial DNA copy numbers and mitochondrial respiration capacity, biogenesis and dynamics). Both a ERK1/2 inhibitor (U0126) and ERK2 siRNA, but not ERK1 siRNA, abolished the protective effect of ATRA against doxorubicin-induced toxicity in H9c2 cells. Remarkably, ATRA did not compromise the anticancer efficacy of doxorubicin in gastric carcinoma cells. ATRA protected cardiomyocytes against doxorubicin-induced toxicity, by activating the ERK2 pathway, without compromising its anticancer efficacy. Therefore, ATRA is a promising candidate as a cardioprotective agent against doxorubicin cardiotoxicity.

Abstract 3490: Topotecan synergizes with CHEK1 inhibitor to induce apoptosis in ovarian cancer cells

Abstract Background Topotecan is a therapeutic option for women with platinum-resistant or -refractory ovarian cancer. However, the dose-limiting toxicity of topotecan is myelosuppression. This led us to seek a combination treatment to augment topotecan anti-cancer activity in a cancer-targeted manner. Ovarian serous cancers, a major subtype, show dysregulated DNA repair pathway and often display a high level of CHEK1, a cell cycle regulator and DNA damage sensor. CHEK1 inhibitors are a novel approach to treatment, and have been used as single agents or in combination chemotherapy in many cancers. Methods We evaluated the cellular effects of topotecan in a panel of high grade serous (HGS) and non-HGS ovarian cancer cells. We then determined IC50s of topotecan in the absence and presence of suboptimal concentrations of CHEK1 inhibitor, PF477736. Synergism between topotecan and PF477736 was calculated based on cellular viability assays. Cytotoxic effect of the combined treatment was compared with apoptotic activities by Caspase3/7 activity assay and Western blotting of cleaved-PARP1 and γH2A.X. Results Non-HGS ovarian cancer cells were generally more sensitive to topotecan treatment compared to HGS ovarian cancer cells. When combined with suboptimal concentrations of CHEK1 inhibitor, topotecan potently and synergistically inhibited the proliferation of HGS ovarian cancer cells. This dramatic synergism in cellular toxicity was consistent with increases in markers of apoptosis. Conclusions Our findings suggest that the addition of CHEK1 inhibitor increases the response of ovarian cancer cells to topotecan. Furthermore, reduced dosages of both drugs achieved maximal cytotoxic effects by combining topotecan with CHEK1 inhibitor. This strategy would potentially minimize side effects of the drugs for extended clinical benefit. Citation Format: Marianne K. H. Kim, Jana James, Christina Annunziata. Topotecan synergizes with CHEK1 inhibitor to induce apoptosis in ovarian cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3490. doi:10.1158/1538-7445.AM2015-3490

Human mesenchymal stem cells are resistant to Paclitaxel by adopting a non-proliferative fibroblastic state.

Human mesenchymal stem cell (hMSC) resistance to the apoptotic effects of chemotherapeutic drugs has been of major interest, as these cells can confer this resistance to tumor microenvironments. However, the effects of internalized chemotherapeutics upon hMSCs remain largely unexplored. In this study, cellular viability and proliferation assays, combined with different biochemical approaches, were used to investigate the effects of Paclitaxel exposure upon hMSCs. Our results indicate that hMSCs are highly resistant to the cytotoxic effects of Paclitaxel treatment, even though there was no detectable expression of the efflux pump P-glycoprotein, the usual means by which a cell resists Paclitaxel treatment. Moreover, Paclitaxel treatment induces hMSCs to adopt a non-proliferative fibroblastic state, as evidenced by changes to morphology, cellular markers, and a reduction in differentiation potential that is not directly coupled to the cytoskeletal effects of Paclitaxel. Taken together, our results show that Paclitaxel treatment does not induce apoptosis in hMSCs, but does induce quiescence and phenotypic changes.

Post-thaw cellular therapy products trypan blue viability assay standardization by dextran40 sample dilution and time-lapse digital microphotography

Post-thaw cellular therapy products trypan blue viability assay standardization by dextran40 sample dilution and time-lapse digital microphotography

Topotecan synergizes with CHEK1 (CHK1) inhibitor to induce apoptosis in ovarian cancer cells.

BackgroundTopotecan (TPT) is a therapeutic option for women with platinum-resistant or -refractory ovarian cancer. However, the dose-limiting toxicity of TPT is myelosuppression. This led us to seek a combination treatment to augment TPT anti-cancer activity in a cancer-targeted manner. Ovarian serous cancers, a major subtype, show dysregulated DNA repair pathway and often display a high level of CHEK1 (CHK1), a cell cycle regulator and DNA damage sensor. CHEK1 inhibitors are a novel approach to treatment, and have been used as single agents or in combination chemotherapy in many cancers.MethodsWe evaluated the cellular effects of TPT in a panel of high grade serous (HGS) and non-HGS ovarian cancer cells. We then determined IC50s of TPT in the absence and presence of CHEK1 inhibitor, PF477736. Synergism between TPT and PF477736 was calculated based on cellular viability assays. Cytotoxic effect of the combined treatment was compared with apoptotic activities by Caspase3/7 activity assay and Western blotting of cleaved-PARP1 and γH2AX.ResultsNon-HGS ovarian cancer cells were generally more sensitive to TPT treatment compared to HGS ovarian cancer cells. When combined with CHEK1 inhibitor, TPT potently and synergistically inhibited the proliferation of HGS ovarian cancer cells. This dramatic synergism in cellular toxicity was consistent with increases in markers of apoptosis.ConclusionsOur findings suggest that the addition of CHEK1 inhibitor increases the response of ovarian cancer cells to TPT. Furthermore, reduced dosages of both drugs achieved maximal cytotoxic effects by combining TPT with CHEK1 inhibitor. This strategy would potentially minimize side effects of the drugs for extended clinical benefit.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1231-z) contains supplementary material, which is available to authorized users.

Overexpression of Nrf2 attenuates Carmustine-induced cytotoxicity in U87MG human glioma cells.

BackgroundMalignant glioma is one of the most devastating tumors in adults with poor patient prognosis. Notably, glioma often exhibits resistance to conventional chemotherapeutic approaches, complicating patient treatments. However, the molecular mediators involved in tumor chemoresistance remain poorly defined, creating a barrier to the successful management of glioma. In the present study, we hypothesized that the antioxidant transcription factor, Nrf2 (nuclear factor erythroid-derived 2 like 2), attenuates glioma cytotoxicity to Carmustine (BCNU), a widely used chemotherapeutic agent known to modulate cellular oxidative balance.MethodsTo test the hypothesis, we employed human malignant glioma cell line, U87MG and overexpression of Nrf2 in glioma cells was achieved using both pharmacological and genetic approaches.ResultsNotably, induction of Nrf2 was associated with increased expression of heme oxygenase-1 (HO-1), a stress inducible enzyme involved in anti-oxidant defense. In addition, over expression of Nrf2 in U87MG cells significantly attenuated the cytotoxicity of Carmustine as evidenced by both cellular viability assay and flow cytometry analysis. Consistent with this, antioxidants such as glutathione and N-acetyl cysteine significantly reduced Carmustine mediated glioma cytotoxicity.ConclusionsTaken together, these data strongly implicate an unexplored role of Nrf2 in glioma resistance to Carmustine and raise the possible use of Nrf2 inhibitors as adjunct to Carmustine for the treatment of malignant glioma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1134-z) contains supplementary material, which is available to authorized users.