Drug-free Medium Research Articles

MicroRNAs as early toxicity signatures of doxorubicin in human-induced pluripotent stem cell-derived cardiomyocytes.

An in depth investigation at the genomic level is needed to identify early human-relevant cardiotoxicity biomarkers that are induced by drugs and environmental toxicants. The main objective of this study was to investigate the role of microRNAs (miRNAs) as cardiotoxicity biomarkers using human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) that were exposed to doxorubicin (DOX) as a “gold standard” cardiotoxicant. hiPSC-CMs were exposed to 156 nM DOX for 2 days or for 6 days of repeated exposure, followed by drug washout and incubation in drug-free culture medium up to day 14 after the onset of exposure. The induced miRNAs were profiled using miRNA microarrays, and the analysis of the data was performed using the miRWalk 2.0 and DAVID bioinformatics tools. DOX induced early deregulation of 14 miRNAs (10 up-regulated and 4 down-regulated) and persistent up-regulation of 5 miRNAs during drug washout. Computational miRNA gene target predictions suggested that several DOX-responsive miRNAs might regulate the mRNA expression of genes involved in cardiac contractile function. The hiPSC-CMs exposed to DOX in a range from 39 to 156 nM did not show a significant release of the cytotoxicity marker lactate dehydrogenase (LDH) compared to controls. Quantitative real-time PCR analyses confirmed the early deregulation of miR-187-3p, miR-182-5p, miR-486-3p, miR-486-5p, miR-34a-3p, miR-4423-3p, miR-34c-3p, miR-34c-5p and miR-1303, and also the prolonged up-regulation of miR-182-5p, miR-4423-3p and miR-34c-5p. Thus, we identified and validated miRNAs showing differential DOX-responsive expression before the occurrence of cytotoxicity markers such as LDH, and these miRNAs also demonstrated the significant involvement in heart failure in patients and animal models. These results suggest that the DOX-induced deregulated miRNAs in human CMs may be used as early sensitive cardiotoxicity biomarkers for screening potential drugs and environmental cardiotoxicants with a similar mechanism of action.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-016-1668-0) contains supplementary material, which is available to authorized users.

Phenotypic Modifications in Staphylococcus aureus Cells Exposed to High Concentrations of Vancomycin and Teicoplanin.

Bacterial cells are known to change the fatty acid (FA) composition of the phospholipids as a phenotypic response to environmental conditions and to the presence of toxic compounds such as antibiotics. In the present study, Staphylococcus aureus cells collected during the exponential growth phase were challenged with 50 and 100 mg/L of vancomycin and teicoplanin, which are concentrations high enough to kill the large majority of the cell population. Colony-forming unit counts showed biphasic killing kinetics, typical for persister cell enrichment, in both antibiotics and concentrations tested. However, fluorescence microscopy showed the existence of viable but non-culturable (VBNC) cells in a larger number than that of possible persister cells. The analysis of the FA composition of the cells showed that, following antibiotic exposure up to 6 h, the survivor cells have an increased percentage of saturated FAs, a significant reduced percentage of branched FAs and an increased iso/anteiso branched FA ratio when compared to cells exhibiting a regular phenotype. This should result in lower membrane fluidity. However, cells exposed for 8–24 h presented an increased branched/saturated and lower iso/anteiso branched FA ratios, and thus increased membrane fluidity. Furthermore, the phenotypic changes were transmitted to daughter cells grown in drug-free media. The fact that VBNC cells presented nearly the same FA composition as those obtained after cell growth in drug-free media, which could only be the result of growth of persister cells, suggest that VBNC and persister phenotypes share the same type of response to antibiotics at the lipid level.

Abstract C106: CBP501 potentiates the appearance of cisplatin-induced indicators of immunogenic cell death and promotes anti-tumor effects in an immuno-competent mouse model

Abstract Introduction: The CBP501 calmodulin-binding peptide is an anti-cancer drug candidate that has completed two Phase II clinical trials for patients with malignant pleural mesothelioma and non-small cell lung carcinoma (NSCLC). CBP501 was previously described as a unique G2 checkpoint-directed agent and as an enhancer of cisplatin (CDDP) uptake. In a post-trial analysis of patients with NSCLC, it was found that overall survival (OS) was prolonged in a subpopulation of patients with normal white blood cell counts (WBC) (WBC < 8000). Here we show that modulation of immunogenic cell death might be a novel function of CBP501. Based on this novel activity of CBP501, combined treatment with PD-1 blocking antibodies in vivo was also explored. Methods: CT26WT, a strain of CBP501 sensitive cells, were treated with clinically achievable dose levels of CDDP (10 or 20 uM) and 0.5 uM CBP501 for 0.75 h in vitro. After exchange with fresh drug-free medium, cells were incubated for 24, 48, or 72 h for IB analysis of phospho-eIF2-alpha, for calreticulin analysis by FACS, or for HMGB1 ELISA. The in vivo mouse study examined the effects of 3 dosing cycles for 3 anti-cancer agents, alone or in different combinations [CDDP: 5 mg/kg x 1/week, CBP501: 7.5 mg/kg x 3/week, anti-mPD1 antibody (RMP1-14): 200 ug x 1/week] using immuno-competent BALB/c mice bearing subcutaneous inoculated CT26 WT cells. Results: In vitro, CDDP in combination with CBP501 elicited increased death in CT26WT cells, as well as an increase in different indicators of immunogenic cell death. These indicators include induction of phospho-eIF2-alpha, increase in cell surface-exposed calreticulin, and extracellular release of HMGB1. In an in vivo BALB/c mouse model, CDDP-treated mice showed a reduction of tumor growth by 52.7% as compared to vehicle-treated mice. CBP501 + CDDP showed an additional reduction of tumor growth by 63.1% as compared to vehicle-treated mice. Treatment with anti-mPD-1 antibody alone showed a slight reduction of tumor growth by 25.2% as compared to vehicle-treated mice. However, combined treatment with anti-mPD-1 + CDDP or anti-mPD-1 + CDDP + CBP501 showed significant reductions in the tumor growth in comparison to the vehicle-treated mice by 69.3% and 78.7%, respectively. Conclusion: These results suggest that the anti-tumor activity of combined CBP501 + CDDP treatment is potentiated by inducing immunogenic cell death. This novel effect might contribute to the prolonged OS found in the phase II clinical trials. Combined treatments that include the anti-mPD-1 immune-checkpoint inhibitor were effective and shall be examined further. Further, immunohistochemical analyses of the effects of this combined treatment on tumor microenvironment are under way. Citation Format: Keiichi Sakakibara, Takuji Sato, Donald W. Kufe, Daniel D. Von Hoff, Takumi Kawabe. CBP501 potentiates the appearance of cisplatin-induced indicators of immunogenic cell death and promotes anti-tumor effects in an immuno-competent mouse model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C106.

Abstract 3854: Inhibition of uracil DNA glycosylase (UDG) sensitizes cancer cells to thymidylate synthase inhibitors

Abstract Thymidylate synthase (TS) is responsible for the de novo synthesis of thymine nucleotides that are used as building blocks for DNA synthesis. Two classes of therapeutic agents inhibit TS including fluropyrimidines (eg, 5-FU, 5-FdU) and antifolates (eg, pemetrexed), which lead to depletion of deoxythymidine triphosphate (dTTP) and expansion of deoxyuridine triphosphate (dUTP). Fluropyrimidines are also metabolized to 5-fluorouridine triphosphate (5FdUTP). As a result, both elevated dUTP and 5FdUTP are incorporated into DNA. Uracil DNA N-glycosylase (UDG or UNG) is the primary DNA repair enzyme to remove uracil and 5-FU bases from DNA and creates abasic (AP) sites, thus initiating base excision repair (BER) pathway. However, whether the cytotoxic effects of TS inhibitors are dependent on UDG activity still remains controversial. Previously, our lab has shown that in DLD1 colon cancer cells, knockout of UDG hypersensitized cells to pemetrexed treatment and the cell death was accompanied by remarkable S phase arrest and stalled replication fork. In this study, we examined the cytotoxicity of both pemetrexed and 5-FdU in SW480, HCT116, DLD1, and HEC1A cancer cell lines after performing stable UDG knockdown (KD) by shRNA. We hypothesized that loss of UDG would sensitize cancer cells to TS inhibitors treatment with accumulation of a large amount of uracil and 5-FU bases in DNA and ultimately lead to replication folk collapse. Results showed that reduced UDG protein and activity levels enhanced the cell killing effect, increased the sub-G1 populations, and delayed cell cycle recovery following TS inhibitors treatment. In response to 5-FdU treatment, cells with UDG shRNA were mainly arrested at late G1 and early S phase by 24 hours of treatment. After releasing cells in drug free medium, loss of UDG displayed delayed recovery from cell cycle arrest in comparison to shRNA scramble control cells. We have also detected a significant induction of the amount of AP sites after incubation of extracted DNA from UDG shRNA KD cells with purified UDG protein following 5-FdU treatment, indicating the accumulation of uracil and 5-FU in DNA as a consequence of 5-FdU treatment. Now, further studies are undergoing to determine the actual levels of uracil and 5-FU incorporation into DNA and the replication status after TS inhibitors treatment. These studies confirm that uracil and 5-FU incorporation due to loss of UDG accelerates stall of replication, collapse of the replication fork, and increased cytotoxicity of TS inhibitors. Citation Format: Yan Yan, Allison Condie, Stanton Gerson. Inhibition of uracil DNA glycosylase (UDG) sensitizes cancer cells to thymidylate synthase inhibitors. [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 3854. doi:10.1158/1538-7445.AM2015-3854

Establishment and characterization of gemcitabine-resistant human cholangiocarcinoma cell lines with multidrug resistance and enhanced invasiveness.

To establish and characterize the gemcitabine-resistant cholangiocarcinoma (CCA) cell lines, CCA KKU‑M139 and KKU‑M214 cell lines were exposed stepwisely to increasing gemcitabine (GEM). The resultant drug-resistant cell lines, KKU‑M139/GEM and KKU‑M214/GEM, retained the resistant phenotype in drug-free medium at least for 2 months. Sulforhodamine B assay demonstrated that KKU‑M139/GEM and KKU‑M214/GEM were 25.88- and 62.31-fold more resistant to gemcitabine than their parental cells. Both gemcitabine-resistant cell lines were cross-resistant to 5-fluorouracil (5-FU), doxorubicin and paclitaxel indicating their multidrug-resistant nature. Using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR and western blot analyses, gemcitabine-resistant cells showed upregulation of RRM1 and downregulation of hENT1 and dCK. In relation to multidrug resistance, these cell lines showed upregulation of multidrug resistance protein 1 (MRP1) leading to an increase of drug efflux. Using cell adhesion and Boyden chamber transwell assays, these cell lines also showed higher cell adhesion, migration and invasion capabilities via the activations of protein kinase C (PKC), focal adhesion kinase (FAK), extracellular signal-regulated kinase-1/2 (ERK1/2) and nuclear factor-κB (NF-κB). Higher activity of matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator (uPA) was also observed by a gelatin zymography assay and a casein-plasminogen zymography assay. Flow cytometry analysis indicated the G2/M arrest regulated by downregulation of cyclin B1 and cyclin-dependent kinase 1 (Cdk1) resulted in an extended population doubling time. Using Annexin V/propidium iodide staining, evasion of apoptosis via an intrinsic pathway was observed in both cell lines in association with upregulation of Bcl-2 and downregulation of Bax. Interestingly, Fas was additionally downregulated in KKU‑M214/GEM supporting the view of its higher GEM resistant characteristics. These findings indicate that long-term exposure of CCA cell lines to gemcitabine induce not only multidrug resistance but also enhance their invasiveness.

Trisomy of chromosome R confers resistance to triazoles in Candida albicans.

Genome plasticity is a hallmark of Candida albicans, and it has been suggested that it generates numerical and structural genomic variations as a means of adaptation. In this study, we used array based comparative genomic hybridization technology and the quantitative real time PCR to investigate the mechanisms by which the following strains obtained by genetic manipulation, CaLY188, CaLY350, CaLY190 and CaLY191, were resistant to antifungal azoles. All four showed trisomy of chromosome R and resistance to azoles. Serial passage of CaLY188 in drug-free medium resulted in chromosome loss, causing chromosome R disomy and loss of azole resistance. Thus we proposed that trisomy of chromosome R contributes to azole resistance.

In vitro embryos production after oocytes treatment with forskolin.

The inhibition of nuclear maturation allows time for the oocyte to accumulate molecules that are important for embryonic development. Thus, the objective of this work was to evaluate the effect of blocking oocyte meiosis with the addition of forskolin, an efficient inhibitor of nuclear maturation, in in vitro maturation (IVM) medium. Forskolin was added to the IVM medium for 6 h at concentrations of 0.1 mM, 0.05 mM or 0.025 mM, then the oocytes were allowed to mature in drug-free medium for 18 h. The oocytes were assessed for the stage of nuclear maturation, the activity and distribution of mitochondria, oocyte ultrastructure, the number of viable cells and the apoptosis rate. After forskolin treatment, the oocytes were fertilized in vitro and cultured for 7 days. On day 7, the blastocyst rate, the ultrastructure, the number of intact cells and the apoptosis rate of the blastocysts were measured. No differences were observed for the stage of nuclear maturation of the oocyte, the mitochondrial activity and distribution, the blastocyst rate or total number of intact cells. However, a higher rate of apoptosis was observed in the blastocysts produced from oocytes blocked for 6 h with the higher concentration of forskolin (P < 0.05). We conclude that all the experimental groups reached the MII stage after the addition of forskolin and that the highest concentration of forskolin caused cellular degeneration without harming embryo production on the 7th day.

Overexpression of MRP4 (ABCC4) and MRP5 (ABCC5) confer resistance to the nucleoside analogs cytarabine and troxacitabine, but not gemcitabine.

We aimed to determine whether the multidrug-resistance-proteins MRP4 (ABCC4) and MRP5 (ABCC5) confer resistance to the antimetabolites cytarabine (Ara-C), gemcitabine (GEM), and the L-nucleoside analog troxacitabine. For this purpose we used HEK293 and the transfected HEK/MRP4 (59-fold increased MRP4) or HEK/MRP5i (991-fold increased MRP5) as model systems and tested the cells for drug sensitivity using a proliferation test. Drug accumulation was performed by using radioactive Ara-C, and for GEM and troxacitabine with HPLC with tandem-MS or UV detection. At 4-hr exposure HEK/MRP4 cells were 2-4-fold resistant to troxacitabine, ara-C and 9-(2-phosphonylmethoxyethyl)adenine (PMEA), and HEK/MRP5i to ara-C and PMEA, but none to GEM. The inhibitors probenecid and indomethacin reversed resistance. After 4-hr exposure ara-C-nucleotides were 2-3-fold lower in MRP4/5 cells, in which they decreased more rapidly after washing with drug-free medium (DFM). Trocacitabine accumulation was similar in the 3 cell lines, but after the DFM period troxacitabine decreased 2-4-fold faster in MRP4/5 cells. Troxacitabine-nucleotides were about 25% lower in MRP4/5 cells and decreased rapidly in MRP4, but not in MRP5 cells. Accumulation of GEM-nucleotides was higher in the MRP4/5 cells. In conclusion: MRP4 and MRP5 overexpression confer resistance to troxacitabine and ara-C, but not to GEM, which was associated with a rapid decline of the ara-C and troxacitabine-nucleotides in HEK/MRP4-5 cells.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-732) contains supplementary material, which is available to authorized users.

Pterocarpans induce tumor cell death through persistent mitotic arrest during prometaphase.

Pterocarpans, a family of isoflavonoids found in the diverse Fabaceae, display potent cytotoxic activity over a panel of tumor cell lines, and among those tested, 2,3,9- trimethoxypterocarpan displays the most potent activity. This study evaluates the effects of 2,3,9-trimethoxypterocarpan and its related derivatives on cell cycle progression and microtubule function in select breast cancer cell lines (MCF7, T47d and HS578T). The pterocarpans, with the exception of 3,4-dihydroxy-9-methoxipterocarpan, induced increased frequencies of mitotic cells by inducing arrest in prometaphase. While microtubule organization in interphase cells was not modified during treatment, mitotic cells exhibited high frequencies of monastral spindles surrounded by condensed chromosomes. Immunofluorescence staining with an anti-γ-tubulin antibody showed double-dot labeling in the spindle polar region, suggesting that pterocarpan treatment blocked centrosome segregation. We found that this mitotic arrest was reversible when the cells were treated for up to 24h followed by recovery in drug-free medium, but not after 48-h treatment followed by incubation in drug-free medium. In that case, treated cells typically underwent cell multinucleation and apoptosis.

An epigenetic mechanism underlying doxorubicin induced EMT in the human BGC-823 gastric cancer cell.

The epithelial to mesenchymal transition (EMT) is a key step during embryonic morphogenesis and plays an important role in drug resistance and metastasis in diverse solid tumors. We previously reported that 48 h treatment of anti-cancer drug doxorubicin could induce EMT in human gastric cancer BGC-823 cells. However, the long term effects of this transient drug treatment were unknown. In this study we found that after 48 h treatment with 0.1 μg/ml doxorubicin, most cells died during next week, while a minor population of cells survived and formed colonies. We propagated the surviving cells in drug free medium and found that these long term cultured drug survival cells (abbreviated as ltDSCs) retained a mesenchymal-like cell morphology, and expressed high levels of EMT-related molecules such as vimentin, twist and β-catenin. The expression of chromatin reprogramming factors, Oct4 and c-myc, were also higher in ltDSCs than parental cells. We further demonstrated that the protein level of p300 was upregulated in ltDSCs, and inhibition of p300 by siRNA suppressed the expression of vimentin. Moreover, the ltDSCs had higher colony forming ability and were more drug resistant when compared to parental cells. Our results suggested that an epigenetic mechanism is involved in the EMT of ltDSCs.

Involvement of HIF-1α activation in the doxorubicin resistance of human osteosarcoma cells.

Osteosarcoma is the most common primary bone cancer in children and adolescents. Despite aggressive treatment regimens, survival outcomes remain unsatisfactory, particularly in patients with metastatic and/or recurrent disease. Unfortunately, treatment failure is commonly due to the development of chemoresistance, for which the underlying molecular mechanisms remain unclear. The aim of the present study was to investigate the role of hypoxia-inducible factor 1α (HIF‑1α) and its signalling pathways as mediators of drug-resistance in human osteosarcoma. Toward this aim, we established two osteosarcoma cell lines selected for resistance to doxorubicin, a drug of choice in the treatment of this tumour. Our results showed that the multidrug resistance (MDR) phenotype was also mediated by HIF-1α, the most important regulator of cell adaptation to hypoxia. Our data showed that this transcription factor promoted the outward transport of intracellular doxorubicin by activating the P-glycoprotein (P-gp) expression in osteosarcoma cells maintained in normoxic conditions. In addition, it hindered doxorubicin-induced apoptosis by regulating the expression of c-Myc and p21. Finally, we observed that the doxorubicin-resistant cells maintained for 2 months of continuous culture in a drug-free medium, lost their drug-resistance and this effect was associated with the absence of HIF-1α expression. The emerging role of HIF-1α in osteosarcoma biology indicates its use as a valuable therapeutic target.

Structural Basis for Inactivation of Giardia lamblia Carbamate Kinase by Disulfiram

Carbamate kinase from Giardia lamblia is an essential enzyme for the survival of the organism. The enzyme catalyzes the final step in the arginine dihydrolase pathway converting ADP and carbamoyl phosphate to ATP and carbamate. We previously reported that disulfiram, a drug used to treat chronic alcoholism, inhibits G. lamblia CK and kills G. lamblia trophozoites in vitro at submicromolar IC50 values. Here, we examine the structural basis for G. lamblia CK inhibition of disulfiram and its analog, thiram, their activities against both metronidazole-susceptible and metronidazole-resistant G. lamblia isolates, and their efficacy in a mouse model of giardiasis. The crystal structure of G. lamblia CK soaked with disulfiram revealed that the compound thiocarbamoylated Cys-242, a residue located at the edge of the active site. The modified Cys-242 prevents a conformational transition of a loop adjacent to the ADP/ATP binding site, which is required for the stacking of Tyr-245 side chain against the adenine moiety, an interaction seen in the structure of G. lamblia CK in complex with AMP-PNP. Mass spectrometry coupled with trypsin digestion confirmed the selective covalent thiocarbamoylation of Cys-242 in solution. The Giardia viability studies in the metronidazole-resistant strain and the G. lamblia CK irreversible inactivation mechanism show that the thiuram compounds can circumvent the resistance mechanism that renders metronidazole ineffectiveness in drug resistance cases of giardiasis. Together, the studies suggest that G. lamblia CK is an attractive drug target for development of novel antigiardial therapies and that disulfiram, an FDA-approved drug, is a promising candidate for drug repurposing.

In vitro Development of Chemotherapy and Targeted Therapy Drug-Resistant Cancer Cell Lines: A Practical Guide with Case Studies

The development of a drug-resistant cell line can take from 3 to 18 months. However, little is published on the methodology of this development process. This article will discuss key decisions to be made prior to starting resistant cell line development; the choice of parent cell line, dose of selecting agent, treatment interval, and optimizing the dose of drug for the parent cell line. Clinically relevant drug-resistant cell lines are developed by mimicking the conditions cancer patients experience during chemotherapy and cell lines display between two- and eight-fold resistance compared to their parental cell line. Doses of drug administered are low, and a pulsed treatment strategy is often used where the cells recover in drug-free media. High-level laboratory models are developed with the aim of understanding potential mechanisms of resistance to chemotherapy agents. Doses of drug are higher and escalated over time. It is common to have difficulty developing stable clinically relevant drug-resistant cell lines. A comparative selection strategy of multiple cell lines or multiple chemotherapeutic agents mitigates this risk and gives insight into which agents or type of cell line develops resistance easily. Successful selection strategies from our research are presented. Pulsed-selection produced platinum or taxane-resistant large cell lung cancer (H1299 and H460) and temozolomide-resistant melanoma (Malme-3M and HT144) cell lines. Continuous selection produced a lapatinib-resistant breast cancer cell line (HCC1954). Techniques for maintaining drug-resistant cell lines are outlined including; maintaining cells with chemotherapy, pulse treating with chemotherapy, or returning to master drug-resistant stocks. The heterogeneity of drug-resistant models produced from the same parent cell line with the same chemotherapy agent is explored with reference to P-glycoprotein. Heterogeneity in drug-resistant cell lines reflects the heterogeneity that can occur in clinical drug resistance.

The isolation of Staphylococcus aureus tea tree oil-reduced susceptibility mutants.

Tea tree oil (TTO)-reduced susceptibility (TTORS) mutants of two Staphylococcus aureus laboratory strains were isolated utilizing TTO gradient plates. Attempts to isolate TTORS mutants employing agar plates containing single TTO concentrations failed. All TTORS mutants demonstrated a small colony variant (SCV) phenotype and produced cells with a smaller diameter, as determined by scanning electron microscopy. The addition of SCV auxotrophic supplements to media did not lead to an increase in TTORS mutant colony size. Revertants were also isolated from the TTORS mutants following growth in drug-free media, and all revertant strains demonstrated phenotypes similar to their respective parent strains. Transmission electron microscopy revealed that an SH1000 TTORS mutant demonstrated a thinner cell wall and novel septal invaginations compared with parent strain SH1000. In addition, comparative genomic sequencing did not reveal any mutations in an SH1000 TTORS mutant previously linked to well-characterized SCV genotypes. This study demonstrates that TTO can select for a unique SCV phenotype.

Modulating autophagy affects neuroamyloidogenesis in an in vitro ischemic stroke model

AimsTo explore the effects of modulating autophagy on neuroamyloidogenesis in an ischemic stroke model of cultured neuroblastoma 2a (N2a)/Amyloid precursor protein (APP)695 cells. MethodsThe ischemic stroke model of N2a/APP695 cells was made by 6h oxygen–glucose deprivation/12h reperfusion (OGDR). Drug administration of 3-methyladenine (3-MA), rapamycin or dl-3-n-butylphthalide (NBP) was started at the beginning of the OGDR and lasted until the end of reperfusion, in order to explore their effects on N2a/APP695 cells under OGDR conditions. Then the cells were incubated in the drug-free and full culture medium under normoxic conditions for 12h. Cell viability and injury were investigated. The key proteins of nuclear factor kappa B (NF-κB) pathway and a key component of autophagy Beclin 1 were detected by Western blotting; immunofluorescence double-staining of amyloid-β (Aβ)1–42 with Beclin 1 was performed to investigate their cellular co-localization relationship; β-secretase and γ-secretase activity assay and Aβ1–42 enzyme-linked immunosorbent assay were performed to investigate the amyloidogenesis. ResultsThe results showed that, OGDR enhanced cell injury, autophagy activity, neuroinflammation and Aβ generation in N2a/APP695 cells; down-regulating autophagy by 3-MA and NBP increased cell viability, decreased lactate dehydrogenase (LDH) production, inhibited the activation of NF-κB pathway, suppressed β- and γ-secretase activities and Aβ generation; while up-regulating autophagy by rapamycin got the opposite results; immunofluorescence double-staining results showed elevated Aβ1–42+ signal was co-localized with Beclin 1+ signal. ConclusionOur data suggested that down-regulating autophagy may inhibit ischemia-induced neuroamyloidogenesis via suppressing the activation of NF-κB pathway. This study might help us to find a new therapeutic strategy to prevent brain ischemic damage and depress the risk of post-stroke dementia.

Comparison of the in vitro Effects of One-day Exposure to Amodiaquine and Praziquantel on Schistosoma mansoni Adult Worm Pairs.

It has been demonstrated that continuous exposure to amodiaquine (AQ) alone elicits in vitro antischistosomal activities at concentrations of 1–10 μg/ml. However, orally administered drugs reach a peak blood concentration within one or two hours and then gradually decrease. The blood concentration does not remain at a constant level over several days as in vitro concentration of continuous drug exposure. In vitro activities by one day exposure to AQ better reflect the actual antischistosomal activities after oral administration than those elicited by continuous exposure.The objective of the present study is to compare the antischistosomal potential of one-day exposure to AQ with that to praziquantel (PZQ), a current antischistosomal drug. Schistosoma mansoni adult worm pairs were incubated with 0 (control), 1, 2, 5 and 10 μg/ml AQ as well as 0.01, 0.02, 0.05 and 0.1 μg/ml PZQ for the first day, and were subsequently incubated in drug-free media for a period of 14 days. The one-day exposure to AQ significantly reduced the daily egg output of the worm pairs at 1–10 μg/ml. The inhibitory effect on egg production continued at 5 and 10 μg/ml but proved temporary at 1 and 2 μg/ml. Furthermore, AQ-induced specific morphological alterations (severe swelling and/or localization of hemozoin) were observed in the worms at 5 and 10 μg/ml. The AQ-specific appearance of the male worms gradually faded during subsequent incubation in drug-free media, although the female worms showed elongation. Meanwhile, PZQ inhibited the egg output of adult worm pairs at concentrations of 0.01–0.1 μg/ml during exposure. The inhibitory effect on egg production continued at 0.05 and 0.1 μg/ml but proved temporary at 0.01 and 0.02 μg/ml. Furthermore, PZQ induced a visible contraction and shortening of the male and female worms at 0.05 and 0.1 μg/ml during exposure, but the PZQ-specific alterations quickly disappeared during subsequent incubation in drug-free media. To our knowledge, this is the first report showing that one-day exposure to AQ inhibits the egg production of adult worm pairs at 1–10 μg/ml and induces specific morphological alterations in the worms at 5 and 10 μg/ml. The present findings have important implications for the evaluation of the therapeutic effects of both AQ monotherapy and combination therapy with artesunate on schistosomiasis in clinical field trials.

A specific inhibitor of CDK1, RO-3306, reversibly arrests meiosis during in vitro maturation of porcine oocytes

CDK1 plays pivotal role in meiotic progression of oocytes from G2 to metaphase II (MII) stage. In this study, we investigated the possibility of utilizing a selective inhibitor of CDK1, RO-3306, as a novel agent for the synchronization of oocyte maturation. Two groups of cumulus-oocyte complexes (COCs) were treated with 10μM RO-3306. The first group was treated for 44h, whereas the second group was transferred to drug-free medium after a 20h treatment. MII-stage oocytes from each group were confirmed by cytoplasmic maturation and embryonic development assays. Treatment of immature porcine oocytes with RO-3306 for 20h arrested them at the germinal vesicle (GV) stage. The GV-arrest effect of RO-3306 was reversible: when RO-3306-arrested COCs were subsequently cultured for 24h in the absence of RO-3306, 76.19±2.68% of these oocytes reached the MII stage after 44h of in vitro maturation, a rate similar to that of non-treated control oocytes (79.08±3.23%). Furthermore, RO-3306-treated oocytes transferred to drug-free media did not differ significantly from controls (P>0.05) with respect to cleavage and blastocyst formation upon parthenogenetic activation. To explore the underlying molecular mechanisms, we examined the expression patterns of four representative maternal transcripts, CDK1, Cyclin B1, GDF9, and BMP15, by real-time polymerase chain reaction (PCR) and poly(A)-test PCR (PAT assay). RO-3306 treatment increased expression of CDK1 but had no effect on the expression of the other genes. These data suggest that RO-3306 efficiently blocks and synchronizes the meiotic progression of porcine oocytes at the GV stage without affecting their meiotic and cytoplasmic maturation.

Induced Dependence On The ALK Kinase Inhibitor Crizotinib In Formerly Sensitive Anaplastic Large Cell Lymphoma Cells

Kinase inhibitors can be highly effective against cancers driven by specific oncogenic kinase proteins, but resistance usually develops after prolonged treatment. Target-dependent mechanisms, typically acquired second-site mutations that prevent drug binding, or target-independent mechanisms, such as downstream or parallel pathway activation, may mediate resistance. Prolonged exposure of kinase-dependent cell lines to inhibitors in vitro to select for resistant populations has identified clinically relevant resistance mechanisms against a number of inhibitors. ALK-positive anaplastic large-cell lymphoma (ALCL) is an uncommon form of peripheral T-cell lymphoma (PTCL) driven by the NPM-ALK fusion kinase, resulting from a t(2;5)(p23;q35) chromosomal rearrangement. ALK inhibitors developed for lung cancer, where a different ALK fusion (EML4-ALK) is found in 6% of cases, have shown promise clinically as a new treatment modality for ALK+ ALCL patients. In this study, we selected three NPM-ALK-dependent ALCL cell lines (Karpas-299, SUP-M2, and SU-DHL-1) for resistance to the ALK inhibitor crizotinib. All three lines initially were highly sensitive the drug (IC50< 100 nM) and required months of exposure at gradually increasing concentrations before acquiring resistance. Previous work has identified specific ALK kinase second-site mutations that promote crizotinib resistance, which we also observed in some resistant lines generated. A unique situation arose, however, in a sub-clone of Karpas-299, that was able to grow in crizotinib at concentrations > 1 µM (Karpas-299CR1000). This line had greatly increased viability in crizotinib at concentrations up to 1 µM, compared to untreated cells, suggesting the drug actually maintained growth. Plated in 250 nM crizotinib, Karpas-299CR1000 grows rapidly, similar to the parent line growing in drug-free media. Plated in drug-free media, however, the line's viability drops rapidly to zero and dies, similar to the parent line plated in 250 nM crizotinib. Therefore the drug that once shut off the signaling necessary for the cells' survival, after prolonged exposure (>9 months), became necessary to maintain it in the sub-clonal population. Melanoma cells driven by BRAF-V600E selected for vemurafenib resistance had a similar phenotype in a recent report (Das Thakur, et al., 2013). These cells amplified mutant BRAF to overcome the inhibitor but came to require the drug to counteract BRAF over-activity with amplification beyond a certain point. Preliminarily, we find a similar effect of NPM-ALK amplification in Karpas-299CR1000 cells but are actively exploring mechanism. In sum, our study shows that long-term exposure of ALK+ ALCL cells to an ALK inhibitor may induce a resistant phenotype that comes to depend on drug presence to prevent over-activity of the mutant kinase. Disclosures:No relevant conflicts of interest to declare.

STAT5 Is a Critical Component Of The Time-Dependent Sensitivity Of CML Cells To TKI Treatment In a Bcr-Abl-Dependent, But JAK2-Independent Manner

IntroductionBcr-Abl1 is necessary and sufficient to cause chronic myeloid leukemia (CML) and as such CML cells are dependent on Bcr-Abl signalling for survival. Targeting CML cells with tyrosine kinase inhibitors (TKIs) commits cells to apoptotic cell death. Bcr-Abl constitutively activates STAT5, however the role of JAK-2 in the activation of STAT5 by Bcr-Abl is controversial. Recent studies of transient Bcr-Abl inhibition indicate that residual low levels of TKI are sufficient to maintain STAT5 inhibition in the absence of sustained Bcr-Abl inhibition. Therefore STAT5 is a highly sensitive measure of kinase activity. We hypothesized that sustained blockade of STAT5 is essential for the commitment of CML cells to apoptosis following inhibition of Bcr-Abl by TKIs. AimTo determine the role of STAT5 and JAK inhibition in the commitment of CML cells to apoptosis. MethodsFactors required for CML cell death were examined in the setting of transient inhibition of Bcr-Abl by TKIs. Induction of apoptosis was assessed by Annexin V/7AAD and the clonogenic potential of CML progenitors assessed by CFU-GM assay. Bcr-Abl and apoptotic signaling pathways were interrogated by western blotting and flow cytometry. Dasatinib was used at 100 nM for potent inhibition of Bcr-Abl. Short term refers to 30 min exposure. Standard washout refers to 3 consecutive washes following potent TKI treatment. Optimal washout refers to 3 washes with 1 h equilibration at 37°C in drug free media between washes. ResultsIn BCR-ABL+ cell lines short term, potent dasatinib exposure followed by optimal washout resulted in reactivation of Bcr-Abl and STAT5, inhibition of apoptosis (83% viable, n=3) and maintenance of colony formation in CML progenitors (CFU-GM: 85% of untreated n=3).Plasma concentrations of dasatinib vary between patients, however peak plasma levels occur up to 6 h after dosing and dasatinib remains available for up to 24 h. CML cell lines and CP-CML CD34+ progenitors were exposed to 100 nM dasatinib for 0.5-8 h before optimal washout. Cell death was achieved if TKI exposure by at least 4 h, with maximal cell death (15% viable, n=3, p=0.008) and reduction of colonies (30.1% of control, p=0.002) achieved after 8 h exposure. Comparison of 30 min and 8 h exposures to 100 nM dasatinib followed by optimal washout was performed to assess the critical signalling components required to induce apoptosis. Reactivation of Bcr-Abl, STAT5 and Erk occurred upon washout following both the 30 min and 8 h exposures, however the 8 h exposure resulted in the inhibition of STAT5 and loss of expression of STAT5 targets Mcl-1 and Bcl-xl, but not Bcl-2. In CP-CML CD34+ cells, prolonged inhibition of STAT5 was observed after 4 h exposure, following optimal washout, highlighting loss of STAT5 activity as potentially critical to irreversible induction of cell death.Continuous inhibition of STAT5 alone with pimozide (Pz) or the specific inhibitor N'-((4-Oxo-4H-chromen-3-yl)methylene)nicotinohydrazide (herein referred to as STAT5i) led to minimal apoptosis (73% and 75% viable, respectively, n=3) when used alone. However, when combined with 30 min exposure to dasatinib (100 nM) STAT5 inhibition proved lethal in a proportion of cells despite optimal washout (57% viable +Pz and 59% +STAT5i). The clonogenic potential CML progenitors was also significantly reduced (12%, p=0.002 and 18% CFU, p=0.003) (Figure 1). [Display omitted] The JAK1/2 kinase inhibitor ruxolitinib was used to assess the involvement of JAK1/2 in Bcr-Abl-dependent activation of STAT5. Similar to the observations with STAT5 inhibition, ruxolitinib had minimal effect on cell death as a sole agent (74% viable). However, in contrast to our observations with STAT5 inhibition, the addition of ruxolitinib to 30 min 100 nM dasatinib exposure did not induce additional cell death (70% viable, p=0.41, n=3). ConclusionSTAT5 is a critical component of the time-dependent sensitivity of CML cells to TKI treatment in a Bcr-Abl-dependent, but JAK-independent manner. In contrast to previous studies describing JAK2 as a promising secondary target for the enhancement of TKI treatment of CML, we demonstrate that inhibition of STAT5 in conjunction with standard TKI therapy is a promising therapeutic strategy for the treatment of CML. Disclosures:Nievergall:CSL: Research Funding. White:Novartis: Research Funding; BMS: Research Funding, Speakers Bureau; Ariad: Research Funding; CSL: Research Funding. Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL: Research Funding.

Survival Pathways Stabilized By Proteasome Inhibition Reveal PIM2 As a Novel Target For Potentiating The Anti-Myeloma Activity Of Carfilzomib

In recent years, new agents for multiple myeloma treatment (e.g., proteasome inhibitors) have become more efficacious, yet nearly all patients eventually relapse and develop refractory disease. Growing evidence suggests that clonal heterogeneity in multiple myeloma may constitute the basis for treatment resistance. Therefore, a multi-pronged approach with novel agents is needed to increase the efficacy of standard therapy and prevent or overcome resistance to standard treatments. We have undertaken a research effort to discover novel targets that potentiate the anti-tumor effects of proteasome inhibition in myeloma cells. We hypothesized that proteins that are stabilized in tumor cells following proteasome inhibition likely constitute components of both pro-apoptotic and pro-survival pathways. A mass spectrometry approach, referred to as UbiScan®, was employed to determine the identity and levels of cellular proteins modified with ubiquitin. MM cell lines (U266 and NCI-H929) were treated with either carfilzomib (CFZ) or bortezomib (BTZ) for 1 hour and the ubiquitome was profiled at 1 and 3 hours after culture in drug-free media. A concentration of 125 nM was chosen in order to reflect physiologically relevant drug and target inhibition levels and to induce cell death in ∼80% of cells after 48 hours. Approximately 400 proteins showed similar increases in ubiquitination with CFZ or BTZ. One of these proteins was PIM2, a serine/threonine proto-oncogene required for plasma cell proliferation that is highly expressed in multiple myeloma cell lines. We determined that ubiquitination on PIM2 was occurring at lysine 61, which is known to be associated with proteasomal degradation. Four hours after exposure to CFZ, PIM2 ubiquitination increased 34.6 and 24.9 fold in U266 and H929 cells, respectively, and similar changes were measured following BTZ treatment. Western blot analysis of CFZ-treated cells showed a dose-dependent accumulation of total PIM2 protein, confirming that the increase in ubiquitination correlated with protein accumulation. Next, we employed a siRNA-mediated knockdown approach to study the role of PIM2 in proteasome inhibitor mediated-cell death. Knockdown of PIM2 caused a 20% - 50% decrease in viability in both myeloma cell lines. When CFZ was added 48 hours after siRNA transfection, a significant and dose-dependent decrease in viability was observed, suggesting a synergistic interaction. Based on these results, we tested the combination of CFZ and (Z)-5-(4-propoxybenzylidene)thiazolidine-2,4-dione (PIM1/2 inhibitor), which is known to inhibit both PIM1 and PIM2. The PIM1/2 inhibitor decreased levels of phosphorylation on 4E-BP1, a downstream target, confirming its activity in cells. Chemical inhibition of PIM2 potentiated the effect of CFZ in both MM cell lines. These data suggest that the combination of targeting PIM2 and the proteasome will be efficacious in the treatment of multiple myeloma. Disclosures: Lin: Onyx Pharmaceuticals, Inc.: Employment. Lowe:Onyx Pharmaceuticals, Inc.: Employment, Equity Ownership. Lerner:Onyx Pharmaceuticals, Inc.: Employment, Equity Ownership. Kirk:Onyx Pharmaceuticals: Employment, Equity Ownership. Arastu-Kapur:Onyx Pharmaceuticals, Inc.: Employment, Equity Ownership.