Drug-treated Cells Research Articles

Abstract 5206: Quantifying DNA-protein crosslinks induced by bifunctional alkylating agents in cultured human leukemia cells

Abstract Bifunctional alkylating agents, such as cisplatin and several nitrosoureas, have clinical utility as antineoplastic drugs largely due to their DNA alkylating and crosslinking activity. Other drug candidates such as laromustine (VNP-40101M) and dianhydrogalacticol (VAL-083) have similar DNA-alkylating activities and have shown success in clinical trials against hard-to-treat cancers such as acute myelogenous leukemia and glioblastoma multiforme. While these agents' interstrand and instrastrand DNA crosslinking activities are widely cited as the critical cytotoxic event, their capacity to crosslink DNA to chromosomally-affiliated proteins may also contribute to their anticancer profile. Indeed, DNA-protein crosslinks (DPCs) are highly toxic phenomena that are difficult for cells to repair and severely compromise gene transcription and DNA replication. Herein, we assess concentration-dependent DPC formation by a wide variety of bifunctional alkylating agents in HL-60 human leukemia cells using two parallel approaches. In one approach, protein-bound DNA was quantified using PicoGreen after precipitating proteins from lysates of drug-treated cells and compared to total DNA concentration. In another, chromosomal DNA was extracted from lysates of drug-treated cells then treated with fluorescein isothiocyanate, which modifies primary amines on proteins. Protein concentration was measured by fluorescence and compared to that of the extracted DNA. The nitrogen mustard mechlorethamine and the chloroethyl nitrosoureas carmustine and lomustine were the most potent agents, yielding significant DPC formation at clinically relevant doses (10−5 M). Cisplatin and laromustine yielded DPCs at similar concentrations, but to a lesser extent. Dianyhydrogalacticol, epichlorohydrin, and diepoxybutane, which all rely on epoxide functional groups for attachment to cellular nucleophiles, required at least an order of magnitude higher drug concentrations for significant DPC observation. These data suggest bifunctional alkylating agents featuring halide leaving groups are most apt to crosslink DNA to nuclear proteins and may offer insights into patterns of cytotoxicity and clinical utility for this important class of chemotherapeuric agents. Citation Format: Hailey L. Cerrato, Sam W. Marchant, Allie H. Naccara, Thomas J. LaJoie, Shawnya Michaels, Dennis Brown, Kevin P. Rice. Quantifying DNA-protein crosslinks induced by bifunctional alkylating agents in cultured human leukemia cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5206.

Abstract 3902: Novel therapeutic targeting of epigenetic aberrations in pediatric sarcomas through combination of ONC201 and HDAC inhibitors

Abstract Patients diagnosed with pediatric sarcomas have poor outcomes with current standard therapy in the setting of relapse, progressive disease, or upfront metastasis. Imipridones, a class of novel small molecules, specifically ONC201, have shown pre-clinical and clinical efficacy in adult malignancies and in brain tumors with aberrant methylation of histone H3 on lysine 27 (associated with H3K27M mutation). ONC201 is an antagonist of dopamine receptor D2 (DRD2), which is a G protein-coupled receptor that is overexpressed in several malignancies. ONC201 inactivates cell proliferation kinases Akt/ERK and induces cell death through the pro-apoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor DR5. Prior in vitro studies of pediatric sarcomas have produced promising data with ERK pathway inhibition, leading to apoptosis and inhibition of metastasis formation. Some pediatric sarcomas, such as malignant peripheral nerve sheath tumors and fusion-positive sarcomas, have been noted to have aberrant activation or modifications of H3K27. H3K27 can be methylated or acetylated and both modifications are abrogated in H3K27M mutated alleles. Changes in histone acetylation and the epigenetic effects of imipridones remain under investigation. We hypothesized that sarcomas with aberrant H3K27 modifications may respond to the novel small molecule similarly to H3K27-mutated brain tumors, and that the combination of ONC201 with histone deacetylase inhibitors (HDACi) could lead to a synergistic effect on cell death mechanisms, potentially through effects of histone acetylation. We performed drug treatment with ONC201 on established pediatric sarcoma cell lines and applied combinatory drug treatment with HDACi, including vorinostat (SAHA). Cell viability was measured after drug treatment in vitro. We investigated markers of cell death in established pediatric sarcoma cell lines via protein analysis and flow cytometry analysis. Using protein quantification studies and downstream target analysis on combination drug-treated cells, we investigated the mechanisms of how these two targeted therapies interact synergistically to cause cell death. We show that ONC201 inactivates cellular proliferation in pediatric sarcomas through the Akt/ERK pathway and induces cell death through the TRAIL pathway. We demonstrated that treatment of cells with ONC201 modified the mitochondrial Clp protease complex, by activating the ClpP protease and by decreasing the chaperone subunit ClpX. Our studies demonstrate that ONC201 induces cell death in pediatric sarcomas, and that ONC201 has potent synergistic effects causing tumor cell death when combined with HDACi. Further studies are underway in elucidating the combinatory epigenetic effects of HDACi and ONC201 in H3K27-aberrant sarcomas, leading to synergistic cell death mechanisms. Our studies present a novel therapeutic combination for pediatric sarcomas that have relapsed or are refractory to standard chemotherapy. Citation Format: Wen-I Chang, Lanlan Zhou, Attila A. Seyhan, Yiqun Zhang, Wafik S. El-Deiry. Novel therapeutic targeting of epigenetic aberrations in pediatric sarcomas through combination of ONC201 and HDAC inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3902.

Drug-induced immune hemolytic anemia investigation: Comparison between tube test and microcolumn agglutination (gel test) for the detection of drug-dependent antibodies in the presence of soluble drug

Testing for drug-dependent antibodies is traditionally performed with the tube method either with drug-treated red blood cells or with untreated red blood cells in the presence of soluble drug. Gel microcolumn agglutination method was compared to tube testing for the demonstration of drug-dependent antibodies in the presence of soluble drug. Patient's samples were tested in parallel by tube and gel microcolumn agglutination method with untreated and/or enzyme-treated red blood cells in the presence of soluble drug. Twenty six different patient's samples were studied and thirty nine tests performed to investigate antibodies directed against fifteen different drugs. There was a good correlation between the results obtained by tube and gel method in terms of analytical sensitivity and specificity. Reactions appeared to be stronger with the gel test than seen with the conventional tube method for most of the drug antibodies investigated. Enzyme-treated cells should be used in addition to untreated cells to improve the sensitivity of the method for detecting drug-dependent antibodies especially those directed against drugs that do not bind firmly to red blood cells. Gel method appeared to be sensitive, reliable, reproducible, and comparable to the conventional tube method for the detection of all the drug-dependent antibodies investigated in this study. Further studies need to be performed to evaluate gel testing for the detection of drug-dependent antibodies that only react with drug-treated red blood cells.

GC–MS based comparative metabolomic analysis of MCF-7 and MDA-MB-231 cancer cells treated with Tamoxifen and/or Paclitaxel

Breast cancer cells MCF-7 and MDA-MB-231 were treated with Tamoxifen (5 μM) or Paclitaxel (1 μM) or with a combination of the two drugs. Herein, we have employed gas chromatography coupled with mass spectroscopy to identify metabolic changes occurring as response to different drug treatments. We report the identification of sixty-one metabolites and overall the two studied cell lines showed a distinct metabolomic profile from each other. Further data analysis indicates that a total of 30 metabolites were significantly differentially abundant in MCF-7 drug-treated cells, most of the metabolic changes occurred when cells were treated with either Tamoxifen (15) or Paclitaxel (25). On the other side, a total of 31 metabolites were significantly differentially abundant in MDA-MB-31 cells with drug treatment. Similarly, to MCF-7 most of the metabolic changes occurred when cells were treated with either Tamoxifen (19) or Paclitaxel (20). In conclusion, this report demonstrates that Tamoxifen and/or Paclitaxel treatment have a pronounced effect on the main metabolic pathways in both breast cancer (BC) cell lines (MCF-7 and MDA-MB231), which could be used as a foundation for future investigations to understand the possible effect of these drugs on different metabolic pathways. SignificanceMetabolic profiling of cancer cells is a promising tool in tumor diagnosis, biomarker discovery and drug treatment protocols, since cancer cells exhibit altered metabolism when compared to normal cells. Although numerous studies have reported the use of various OMICs applications to investigate breast cancer cells, very few of these have performed thorough screening of metabolites in such cells. Our investigation highlights the first study to characterize MCF7 and MDA-MB-231 cancer cells treated with Tamoxifen and/or Paclitaxel and to identify the affected metabolic pathways. Such findings might play an important role in revealing the molecular bases of the underlying mechanism of action of these two frontline anti-breast cancer drugs.

Fast intelligent cell phenotyping for high-throughput optofluidic time-stretch microscopy based on the XGBoost algorithm.

.Significance: The use of optofluidic time-stretch flow cytometry enables extreme-throughput cell imaging but suffers from the difficulties of capturing and processing a large amount of data. As significant amounts of continuous image data are generated, the images require identification with high speed.Aim: We present an intelligent cell phenotyping framework for high-throughput optofluidic time-stretch microscopy based on the XGBoost algorithm, which is able to classify obtained cell images rapidly and accurately. The applied image recognition consists of density-based spatial clustering of applications with noise outlier detection, histograms of oriented gradients combining gray histogram fused feature, and XGBoost classification.Approach: We tested the ability of this framework against other previously proposed or commonly used algorithms to phenotype two groups of cell images. We quantified their performances with measures of classification ability and computational complexity based on AUC and test runtime. The tested cell image datasets were acquired from high-throughput imaging of over 20,000 drug-treated and untreated cells with an optofluidic time-stretch microscope.Results: The framework we built beats other methods with an accuracy of over 97% and a classification frequency of . In addition, we determined the optimal structure of training sets according to model performances under different training set components.Conclusions: The proposed XGBoost-based framework acts as a promising solution to processing large flow image data. This work provides a foundation for future cell sorting and clinical practice of high-throughput imaging cytometers.

LINCS dataset-based repositioning of rosiglitazone as a potential anti-human adenovirus drug

Human adenoviruses (HAdVs) often cause mild respiratory infections. These infections, however, can potentially become fatal in immunosuppressive patients. Unfortunately, there has been no specific anti-HAdV drug approved for treatment of HAdV infections. In this study, a time-course transcriptome of HAdV-infected human lung epithelial cells (A549 cells) was performed and compared with perturbation datasets of 890 drug-treated A549 cells from the library of integrated network-based cellular signatures (LINCS) database to predict previously unknown therapeutic drug-HAdV relationships using a characteristic direction (CD) algorithm. We performed experiments to validate a prediction for the anti-diabetic drug rosiglitazone as a candidate drug for treatment of anti-HAdV both in vivo and in vitro. The Type I interferon (IFNs) signaling pathway was negatively regulated during the course of HAdV infection and rosiglitazone increased STAT1 phosphorylation for antiviral IFN response induction. Taken together, this study confirmed the prospect for re-exploitation of this FDA-approved drug as a potential therapeutic for HAdV infections.

Transient inhibition of rDNA transcription in donor cells improves ribosome biogenesis and preimplantation development of embryos derived from somatic cell nuclear transfer

Ribosomal DNA (rDNA) transcription is a limiting step in ribosome biogenesis, crucial for protein synthesis and cell growth-especially at the early stages of embryonic development-and is regulated in a mammalian target of rapamycin (mTOR)-dependent manner. Our previous report demonstrated that treatment with mTOR inhibitors during artificial embryonic activation improved the development of embryos derived from somatic cell nuclear transfer (SCNT). We hypothesize that inhibition of ribosome biogenesis in somatic cells facilitates reactivation of embryonic nucleolar establishment and ribosome biogenesis in SCNT embryos. Herein, we show that mTOR inhibitors suppressed ribosome biogenesis in somatic cells, and more importantly, improved development potential of SCNT embryos (blastocyst rate, 34% vs 24%). SCNT embryos derived from drug-treated somatic cells exhibited higher levels of 47S, 18S, and 5S rRNAs, upstream binding factor (UBF) mRNA, ribosomal protein S6; they also improved the rebuilding of the nucleolar ultrastructure. In addition, treatment of donor cells with the RNA polymerase I (Pol I) inhibitor cx5461 caused similar effects on SCNT embryos. These results indicated that transient inhibition of rDNA transcription in donor cells facilitated the establishment of functional nucleoli and improved preimplantation development of SCNT embryos.

Label-free characterization of different kinds of cells using optoelectrokinetic-based microfluidics.

We report a novel, to the best of our knowledge, method to rapidly characterize different kinds of cells and drug-treated cancer cells using a label-free biomarker of self-rotation in an optoelectrokinetics (OEK)-based microfluidic platform. OEK incorporates optics and electrokinetics into microfluidics, thereby offering a contact-free, label-free, and rapid approach to the cellular manipulation community. Self-rotational behaviors of four different kinds of cells were experimentally investigated by the frequency-sweeping of an AC bias potential in an optically induced nonuniform and irrotational electric field. The results revealed that these kinds of cells displayed a Gaussian distribution versus the AC frequency as well as different self-rotational speeds under the same conditions. Furthermore, the peak self-rotational speed varied from one kind of cell to another, with that of cancer cells higher than that of normal cells. In addition, MCF-7 cells treated by various concentrations of drug showed remarkably different self-rotational speeds. This finding suggests a high potential of developing a new label-free biomarker to rapidly distinguish different kinds of cancer cells and quantitatively monitor the response of cancer patients to various treatments.

L-cystine-linked BODIPY-adsorbed monolayer MoS2 quantum dots for ratiometric fluorescent sensing of biothiols based on the inner filter effect

This study fabricated a dual-emission probe consisting of monolayer MoS2 quantum dots (M − MoS2 QDs) and L-cystine-linked boron-dipyrromethene (L-Cys-BODIPY) molecules for ratiometric sensing of biothiols, thiol product-related enzyme reactions, and ratiometric imaging of glutathione (GSH)-related reactions in HeLa cells. The formation of L-Cys-BODIPY-adsorbed M − MoS2 QDs (named as BODIPY-M-MoS2 QDs) was demonstrated by comparing them with M − MoS2 QDs using transmission electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. The BODIPY-M-MoS2 QDs exhibited dual-emission bands, excellent biocompatibility, and good resistance to photobleaching. It was found that the adsorbed L-Cys-BODIPY molecules rarely quenched the fluorescence of M − MoS2 QDs, and meanwhile, they were self-quenched by π-π stacking between each BODIPY backbones. The presence of biothiols induced the reduction of weakly fluorescent L-Cys-BODIPY to strongly fluorescent of L-cysteine-conjugated BODIPY. Since having a much higher molar absorption coefficient than L-Cys-BODIPY, the liberated L-cysteine-conjugated BODIPY behaved as an effective inner filter to absorb the excitation light and subsequently quenched the fluorescence of M − MoS2 QDs. The appearance of L-cysteine-conjugated BODIPY could barely affected to the fluorescence lifetime of M − MoS2 QDs, confirming the inner filter effect of L-cysteine-conjugated BODIPY onto the fluorescence of M − MoS2 QDs. The present probe not only provided a linear ratiometric response to 1–10 mM GSH, 1–10 μM cysteine, and 1–10 μM of homocysteine but also remarkably showed the ratiometric detection of thiol products from the reactions of 1–900 units L−1 S-adenosylhomocysteine (SAH) hydrolase and SAH as well as 1–850 units L−1 GSH reductase and disulfide GSH. Additionally, the present probe was well-suited for ratiometric imaging of intracellular GSH levels in non-treated and drug-treated HeLa cells.

Super-Enhancer Redistribution as a Mechanism of Broad Gene Dysregulation in Repeatedly Drug-Treated Cancer Cells

Cisplatin is an antineoplastic drug administered at suboptimal and intermittent doses to avoid life-threatening effects. Although this regimen shortly improves symptoms in the short term, it also leads to more malignant disease in the long term. We describe a multilayered analysis ranging from chromatin to translation-integrating chromatin immunoprecipitation sequencing (ChIP-seq), global run-on sequencing (GRO-seq), RNA sequencing (RNA-seq), and ribosome profiling-to understand how cisplatin confers (pre)malignant features by using a well-established ovarian cancer model of cisplatin exposure. This approach allows us to segregate the human transcriptome into gene modules representing distinct regulatory principles and to characterize that the most cisplatin-disrupted modules are associated with underlying events of super-enhancer plasticity. These events arise when cancer cells initiate without ultimately ending the program of drug-stimulated death. Using a PageRank-based algorithm, we predict super-enhancer regulator ISL1 as a driver of this plasticity and validate this prediction by using CRISPR/dCas9-KRAB inhibition (CRISPRi) and CRISPR/dCas9-VP64 activation (CRISPRa) tools. Together, we propose that cisplatin reprograms cancer cells when inducing them to undergo near-to-death experiences.

약물처리된 심장세포의 세포 수축력 측정을 위한 병렬 폴리머 캔틸레버 제작

약물처리된 심장세포의 세포 수축력 측정을 위한 병렬 폴리머 캔틸레버 제작

Machine Learning in Mass Spectrometry: A MALDI-TOF MS Approach to Phenotypic Antibacterial Screening.

Machine learning techniques can be applied to MALDI-TOF mass spectral data of drug-treated cells to obtain classification models which assign the mechanism of action of drugs. Here, we present an example application of this concept to the screening of antibacterial drugs that act at the major bacterial target sites such as the ribosome, penicillin-binding proteins, and topoisomerases in a pharmacologically relevant phenotypic setting. We show that antibacterial effects can be identified and classified in a label-free, high-throughput manner using wild-type Escherichia coli and Staphylococcus aureus cells at variable levels of target engagement. This phenotypic approach, which combines mass spectrometry and machine learning, therefore denoted as PhenoMS-ML, may prove useful for the identification and development of novel antibacterial compounds and other pharmacological agents.

Androgenic effects on ventricular repolarization: A translational study from the international pharmacovigilance database to iPSC-cardiomyocytes

BackgroundMale hypogonadism, arising from a range of etiologies including androgen-deprivation therapies (ADTs), has been reported as a risk factor for acquired long-QT syndrome (aLQTS) and torsades de pointes (TdP). A full description of the clinical features of aLQTS associated with ADT and of underlying mechanisms is lacking. MethodsWe searched the international pharmacovigilance database VigiBase for men (n=6 560 565 individual case safety reports) presenting with aLQTS, TdP, or sudden death associated with ADT. In cardiomyocytes derived from induced pluripotent stem cells from men, we studied electrophysiological effects of ADT and dihydrotestosterone. ResultsAmong subjects receiving ADT in VigiBase, we identified 184 cases of aLQTS (n=168) and/or TdP (n=68; 11% fatal), and 99 with sudden death. Of the 10 ADT drugs examined, 7 had a disproportional association (reporting odds ratio=1.4–4.7; P<0.05) with aLQTS, TdP, or sudden death. The minimum and median times to sudden death were 0.25 and 92 days, respectively. The androgen receptor antagonist enzalutamide was associated with more deaths (5430/31 896 [17%]; P<0.0001) than other ADT used for prostate cancer (4208/52 089 [8.1%]). In induced pluripotent stem cells, acute and chronic enzalutamide (25μM) significantly prolonged action potential durations (action potential duration at 90% when paced at 0.5Hz; 429.7±27.1 (control) versus 982.4±33.2 (acute, P<0.001) and 1062.3±28.9ms (chronic; P<0.001), and generated afterdepolarizations and/or triggered activity in drug-treated cells (11/20 acutely and 8/15 chronically). Enzalutamide acutely and chronically inhibited delayed rectifier potassium current, and chronically enhanced late sodium current. Dihydrotestosterone (30nM) reversed enzalutamide electrophysiological effects on induced pluripotent stem cells. ConclusionQT prolongation and TdP are a risk in men receiving enzalutamide and other ADTs. Clinical trial registrationURL: https://www.clinicaltrials.gov. Unique identifier: NCT03193138.

Abstract P5-05-14: Cyclin-dependent kinases inhibitors improve antimetabolite drug potency depending on SAMHD1 expression

Abstract Introduction Cyclin-dependent kinase (CDK) 4/6 inhibitors represent a new paradigm in the treatment of hormone receptor (HR) positive and HER-2 negative metastatic breast cancer (MBC). However, despite their evident clinical benefit, the cellular and molecular mechanisms underlying treatment efficacy and drug failure are still largely unknown. CDK4/6 inhibitors block the cyclin D1-CDK4/6 interaction, leading to inhibition of cell cycle progression through the G1/S phase and inhibiting cell proliferation. SAMHD1 is a dNTPase that maintains the intracellular dNTP pool at levels adequate for DNA replication and repair. SAMHD1 function is regulated by phosphorylation, a process controlled by CDKs. Moreover, SAMHD1 has also been shown to modify the efficacy of different nucleotide analogues. Here, we aim to evaluate the capacity of selective CDK4/6 inhibitors to modify the efficacy of different antimetabolites currently used for cancer treatment. Material and methods: Cytotoxic activity of 5-fluorouracil, its prodrug capecitabine and the anti-folate pemetrexed was evaluated alone or in combination with the CDK4/6 inhibitor palbociclib in breast cancer cell lines (T47D and MDA-MB-468) and primary cells expressing or not SAMHD1 through HIV-2 Vpx mediated degradation. Cytotoxicity was measured either by colorimetric tetrazolium dye MTT method or flow cytometry. Drug combinations were evaluated by calculating the combination index (CI) through the ixobologram equation, considering a synergic drug combination a CI &amp;lt;1. Intracellular dNTP content was determined using a polymerase- based method. Expression of relevant genes was measured by qPCR mRNA detection. SAMHD1 expression and phosphorylation were measured by Western blot. Results Analysis of cell cycle profile and protein expression of drug-treated cells confirmed that CDK4/6 control SAMHD1 phosphorylation and decrease intracellular dNTPs. CDK4/6 inhibitors palbociclib, ribociclib and abemaciclib blocked SAMHD1 phosphorylation, whereas SAMHD1 protein expression was not affected. In addition, we observed a concomitant dephosphorylation and decreased expression of Rb, the natural substrate of CDK6, and a known marker of CDK4/6 inhibitor activity. In combination, palbociclib enhanced the efficacy of pemetrexed or 5-fluorouracil up to 6-fold, indicating strong synergy (CI=0,0049-0,404). Interestingly, the synergistic effect was lost in cells not expressing SAMHD1. Palbociclib did not change antimetabolite efficacy in the breast cancer MDA-MB-468 cell line. However, Rb and pRb were not detected in MDA-MB-468 cells by mRNA qPCR or Western blot. Conclusion CDK4/6 inhibitors induce the activation of SAMHD1. Taken together, our results suggest that CDK4/6 inhibitor-mediated enhancement of antimetabolite activity is dependent on the regulation of SAMHD1 function. CDK4/6 inhibitors restrict cells in G1 but also prevent SAMHD1 phosphorylation that sensitizes tumoral cells to antimetabolite drugs. Thus, pharmacological modulation of SAMHD1 activity has the potential to improve the efficacy of cancer therapies and paves the way to the identification of malignancies that may be treated with new drug combinations. Combinations with selective CDK4/6 inhibitors plus pemetrexed or capecitabine could be effective in the treatment of cancer after first line drug-failure. SAMHD1 could be a useful biomarker to predict treatment response. Citation Format: Eudald Felip, Roger Badia, Mireia Margelí, Marc Castellví, Vanesa Quiroga, Iris Teruel, Beatriz Cirauqui, Margarita Romeo, Ifeanyi Jude Ezeonwumelu, Eva Riveira-Muñoz, José Esté, Ester Ballana. Cyclin-dependent kinases inhibitors improve antimetabolite drug potency depending on SAMHD1 expression [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-14.

Morin Inhibits Ovarian Cancer Growth through the Inhibition of NF-κB Signaling Pathway.

Ovarian cancer has the highest mortality in gynecological tumors without effective therapeutic drugs as a result of drug-resistance for long-term utilization. Morin has been reported to possess powerful anti-tumor effects in several cancers. The present study aims to investigate whether Morin could influence ovarian cancer growth and underlying mechanisms. Morin was administered to cultured cells in vitro and formed tumors in vivo. MTT and colony formation assays were performed to explore the effects of Morin on the proliferation and colony formation of OVCAR3 and SKOV3 ovarian cancer cells. Western blot, RT-qPCR, immunofluorescence as well as ELISA were used to detect protein and mRNA expression of target factors. Tumor formation was performed to investigate tumorigenesis ability of drug-treated cells. The proliferation and colony size of OVCAR3 and SKOV3 were significantly decreased after Morin administration. The expression of NF-κB and inflammatory cytokine IL6/8 induced by TNF-α can be inhibited by Morin. Furthermore, Morin inhibited the volume of ovarian cancer tumors in nude mice. Morin effectively alleviates ovarian cancer growth, inhibits the inflammatory response, and reduces tumor size via modulation of the NF-κB pathway.

Olaparib Potentiates Anticancer Drug Cytotoxicity via 53BP1 in Oesophageal Squamous Cell Carcinoma Cells.

Olaparib was previously shown to synergistically enhance the cytotoxicity of DNA synthesis inhibitors in oesophageal carcinoma (OC) cell lines. However, the mechanisms of this synergy are not fully understood. As P53 binding protein 1 (53BP1) expression was previously shown to potentiate the anticancer effect of olaparib, we investigated the involvement of 53BP1 in the synergetic cytotoxic effects of olaparib and anticancer drugs in KYSE70 cells. Experiments included small interfering RNA transfection, growth inhibition assays, western blots, immunofluorescence, and flow cytometry. The toxicity of DNA synthesis-inhibiting agents plus olaparib was decreased when 53BP1 was depleted. Olaparib cotreatment significantly increased phosphorylated H2A histone family member X (γH2AX) foci as well as 53BP1/γH2AX co-localisation in anticancer drug-treated cells. Silencing of 53BP1 suppressed anticancer drug-induced apoptosis with or without olaparib. Olaparib potentiates the cytotoxicity of anticancer drugs through 53BP1 in OC cells.

Mapping of biomechanical properties of cell lines on altered matrix stiffness using atomic force microscopy.

Understanding nanomechanical properties such as elastic modulus, cell adhesion force and stiffness of healthy-, cancerous- and drug-treated cancer cells under varying matrix microenvironment have great roles in cellular processes like morphogenesis, mechanotransduction, focal adhesion, motility, metastasis and drug delivery. AFM is an important technique to depict nanomechanical properties and mechanoadaptation response of cell lines in different microenvironment. In the present study, two different cell categories, i.e. healthy and cancerous, having two different types in each category, i.e. 3T3 fibroblast, HaCaT keratinocyte, untreated and drug-treated MCF-7 and MDA-MB 231 were chosen for cell culture on PDMS matrix of different stiffness without any prior protein layer to facilitate unbiased cell growth. Subsequently cell mechanics response was evaluated by AFM technique and correlated with immunofluorescence assay. Furthermore, cell culture was performed to stiffer matrix after initial incubation on soft matrix and analysed the cell mechanics using AFM and fluorescence assay to understand cell adaptation with matrix stiffness. Results demonstrate deviations in elastic modulus, stiffness and adhesion force induced by matrix stiffness in all four cell lines. Study revealed the doxorubicin drug can significantly influence the cell mechanics and disease state of cancer cells. Variation of cytoskeleton structure, actin fibre and focal adhesion primarily accounts for cell mechanics response in terms of elastic modulus, stiffness and adhesion of cell under varying matrix stiffness. Experimental evidence demonstrates that a greater understanding of the mechanics in terms of cellular deformability and its interactions with extracellular environments offer enormous potential for new advances in disease diagnostics, therapeutics and influence of drug treatment in several complex processes.

Intelligent frequency-shifted optofluidic time-stretch quantitative phase imaging.

Optofluidic time-stretch quantitative phase imaging (OTS-QPI) is a powerful tool as it enables high-throughput (>10,000 cell/s) QPI of single live cells. OTS-QPI is based on decoding temporally stretched spectral interferograms that carry the spatial profiles of cells flowing on a microfluidic chip. However, the utility of OTS-QPI is troubled by difficulties in phase retrieval from the high-frequency region of the temporal interferograms, such as phase-unwrapping errors, high instrumentation cost, and large data volume. To overcome these difficulties, we propose and experimentally demonstrate frequency-shifted OTS-QPI by bringing the phase information to the baseband region. Furthermore, to show its boosted utility, we use it to demonstrate image-based classification of leukemia cells with high accuracy over 96% and evaluation of drug-treated leukemia cells via deep learning.

Cannabinoid Signaling in Glioma Cells.

Cannabinoids are a group of structurally heterogeneous but pharmacologically related compounds, including plant-derived cannabinoids, synthetic substances and endogenous cannabinoids, such as anandamide and 2-arachidonoylglycerol. Cannabinoids elicit a wide range of central and peripheral effects mostly mediated through cannabinoid receptors. There are two types of specific Gi/o-protein-coupled receptors cloned so far, called CB1 and CB2, although an existence of additional cannabinoid-binding receptors has been suggested. CB1 and CB2 differ in their predicted amino acid sequence, tissue distribution, physiological role and signaling mechanisms. Significant alterations of a balance in the cannabinoid system between the levels of endogenous ligands and their receptors occur during malignant transformation in various types of cancer, including gliomas. Cannabinoids exert anti-proliferative action in tumor cells. Induction of cell death by cannabinoid treatment relies on the generation of a pro-apoptotic sphingolipid ceramide and disruption of signaling pathways crucial for regulation of cellular proliferation, differentiation or apoptosis. Increased ceramide levels lead also to ER-stress and autophagy in drug-treated glioblastoma cells. Beyond blocking of tumor cells proliferation cannabinoids inhibit invasiveness, angiogenesis and the stem cell-like properties of glioma cells, showing profound activity in the complex tumor microenvironment. Advances in translational research on cannabinoid signaling led to clinical investigations on the use of cannabinoids in treatments of glioblastomas.

Development of a high-throughput screen to identify small molecule enhancers of sarcospan for the treatment of Duchenne muscular dystrophy

BackgroundDuchenne muscular dystrophy (DMD) is caused by loss of sarcolemma connection to the extracellular matrix. Transgenic overexpression of the transmembrane protein sarcospan (SSPN) in the DMD mdx mouse model significantly reduces disease pathology by restoring membrane adhesion. Identifying SSPN-based therapies has the potential to benefit patients with DMD and other forms of muscular dystrophies caused by deficits in muscle cell adhesion.MethodsStandard cloning methods were used to generate C2C12 myoblasts stably transfected with a fluorescence reporter for human SSPN promoter activity. Assay development and screening were performed in a core facility using liquid handlers and imaging systems specialized for use with a 384-well microplate format. Drug-treated cells were analyzed for target gene expression using quantitative PCR and target protein expression using immunoblotting.ResultsWe investigated the gene expression profiles of SSPN and its associated proteins during myoblast differentiation into myotubes, revealing an increase in expression after 3 days of differentiation. We created C2C12 muscle cells expressing an EGFP reporter for SSPN promoter activity and observed a comparable increase in reporter levels during differentiation. Assay conditions for high-throughput screening were optimized for a 384-well microplate format and a high-content imager for the visualization of reporter levels. We conducted a screen of 3200 compounds and identified seven hits, which include an overrepresentation of L-type calcium channel antagonists, suggesting that SSPN gene activity is sensitive to calcium. Further validation of a select hit revealed that the calcium channel inhibitor felodipine increased SSPN transcript and protein levels in both wild-type and dystrophin-deficient myotubes, without increasing differentiation.ConclusionsWe developed a stable muscle cell line containing the promoter region of the human SSPN protein fused to a fluorescent reporter. Using the reporter cells, we created and validated a scalable, cell-based assay that is able to identify compounds that increase SSPN promoter reporter, transcript, and protein levels in wild-type and dystrophin-deficient muscle cells.