Senna velutina leaves and to assess its antioxidant and cytotoxic activities in leukemic cells. The antioxidant properties were evaluated using a DPPH free radical scavenging assay and by examining the extract's inhibition of AAPH-induced lipid peroxidation in human erythrocytes. Its cytotoxicity and possible mechanisms of action were assessed in Jurkat and K562 leukemic cell lines. The ethanol extract contained flavonoids, such as epigallocatechin, epicatechin, kaempferol heteroside, rutin, and dimeric and trimeric proanthocyanidin derivatives. The extract exhibited antioxidant activity by scavenging free radicals and antihemolytic action, and it decreased malondialdehyde content in human erythrocytes. Furthermore, the extract also induced leukemic cell death by activating intracellular calcium and caspase-3, decreasing mitochondrial membrane potential, and arresting the cell cycle in S and G2 phases. Hence, S. velutina leaf extract contains antioxidant and antileukemic biomolecules with potential applications in diseases associated with oxidative stress and in the inhibition of tumor cell proliferation.Natural products can be a source of biomolecules with antioxidant activity which are able to prevent oxidative stress-induced diseases and show antitumor activity, making them important sources of new anticancer drug prototypes. In this context, this study aimed to analyze the chemical composition of an ethanol extract of Several diseases, including cancer, diabetes, atherosclerosis, inflammatory diseases, and premature aging, are related to oxidative stress . OxidatiCancer is an oxidative stress-related disease that causes high rates of morbidity and mortality in the global population . LeukemiBiomolecules with anticancer activity at low therapeutic doses and with reduced side effects have been increasingly sought in recent decades . Between Senna genus has been used in Brazilian folk medicine for its antioxidant, antimicrobial −, was based on the fragmentation pattern of epigallocatechin proposed by Dou et al. [ m/z 261 [M-CO2]−, m/z 219 [M-C4H6O2]−, m/z 179 [M-C6H6O3]−, m/z 167 [M-C7H6O3]−, and m/z 165 [M-C7H8O3]−, and also on the detection of UV absorption at 270 nm.The identification of compound 2, withu et al. , namely, m/z 285 [C15H9O6]−. Several compounds of this class have been described in Senna genus [Compounds 4, 8, and 9 showed UV absorption patterns that were characteristic of flavonols (270 and 340 nm). Their fragmentation patterns were consistent with those of heteroside derivatives of kaempferol, whose main fragment consists ofna genus , 24. Senna genus; some authors consider them to be chemical markers for the genus [Dimeric  and trimeric (compounds 15–22) proanthocyanidins, comprising cassiaflavan, afzelechin, epicatechin, epigallocatechin, and naringenin subunits, were also observed. These derivatives, although rare in nature, are often reported inhe genus –27. The he genus . Fragmenhe genus . A complhe genus , 30. Thu50 and maximum activity values relative to ascorbic acid; however, these values were lower than those obtained with BHT . ESV was able to protect erythrocytes for 5 h over the tested 50–125 on range , demonstμg/mL, the MDA levels were decreased throughout the course of the assay (data not shown) and after 5 h of incubation  (Figures μg/mL of extract, respectively (Figures ESV promoted cell death in both tested cell lines. IC Figures . ESV tre Figures .μg/mL of extract, respectively (Figures The mitochondrial membrane potentials of Jurkat and K562 leukemic cells decreased after 24 h of incubation with ESV, as evidenced by a decrease in red fluorescence and an increase in green fluorescence compared to untreated cells. The mitochondrial membrane potential was reduced by 91.0 ± 4.3% in Jurkat cells and by 74.7 ± 7.3% in K562 cells after treatment with 27.6 and 67.5  Figures .The Jurkat cell line, which was more sensitive to ESV activity, was used to investigate ESV-promoted cell death mechanisms. The fluorescence histogram  showed a2+ chelator BAPTA-AM partially inhibited ESV-induced death in Jurkat cells (Jurkat cells were preincubated with the pan-caspase inhibitor Z-VAD-FMK to assess whether ESV cytotoxicity was mainly mediated by caspase activation. Z-VAD-FMK pretreatment did not decrease ESV-induced cell death . Howeverat cells .Histograms were used to show the distributions of cell cycle phases in control and ESV-treated Jurkat cells after 24 h of incubation . The resBrazilian biodiversity is rich in active compounds with high potential for development of new therapeutic drugs, particularly antioxidants and anticancer agents. Several plant species found in Brazil have been characterized for their antioxidant and cytotoxic activities in several tumor cell lines –33. S. velutina leaves exhibited antioxidant activity and showed cytotoxic effects against two leukemic cell lines, Jurkat and K562. The antioxidant activity of ESV was demonstrated in AAPH-incubated human erythrocytes; DPPH free radical scavenging and inhibition of lipid peroxidation led to decreases in oxidative hemolysis and malondialdehyde production. This activity of ESV is likely related to the presence of flavone derivatives with antioxidant activity, such as epigallocatechin, epicatechin, rutin, kaempferol glycosides, and dimeric and trimeric proanthocyanidins, in the leaves [In the present study, an ethanol extract ofe leaves –37.Flavonoids can donate hydrogen atoms to radicals, protecting against lipid peroxidation, and this ability is associated with the presence of a dihydroxylated B-ring . SimilarExcess free radicals in the body can promote not only lipid peroxidation but also oxidative DNA damage, leading to the development of early stages of mutagenesis and carcinogenesis , 41. ThuThe cytotoxic activity of ESV against leukemic lines was evaluated with an Annexin/PI-stain cell death assay. Furthermore, activation of caspase-3 was observed but was not confirmed to be a main mechanism of cell death when the pan-caspase inhibitor Z-VAD-FMK was used. Different extracts can promote several different death mechanisms simultaneously because of their different compositions. This fact complicates the analysis and identification of specific cell death pathways.The ESV-induced decrease in mitochondrial membrane potential is consistent with the observed cytotoxic effect of ESV against leukemic cells. The changes in potential are likely due to increased mitochondrial membrane permeability arising from an increase in intracellular calcium levels, a characteristic of necrotic cell death , 43.The present study demonstrated the involvement of calcium in cell death, as the reduced cell viability of extract-treated cells was reversed by incubation with the calcium chelator BAPTA-AM. High calcium levels promote the opening of mitochondrial permeability transition pores; these pores are nonselective and thus release the contents of the intermembrane space of the mitochondrion .Another mechanism of the ESV-induced cytotoxicity against Jurkat cells consists of cell cycle arrest. ESV promoted a decrease in the number of cells in G0/G1 phase and an increase in the number of cells in S and G2 phases. Established anticancer drugs, such as cisplatin and doxorubicin, exert a similar profile change in the tumor cell cycle , 45. Mue S. velutina leaves exhibited antioxidant activity and showed cytotoxic effects on leukemic cells by activating intracellular calcium and caspase-3, decreasing mitochondrial membrane potential, and arresting the cell cycle in S and G2 phases.In conclusion, an ethanol extract of


There are number of studies which report that BCL-2 anti-apoptotic proteins  are highly expressed in cervical cancer tissues compared to the normal cervical epithelia. Despite these reports, targeting these proteins for cervical cancer treatment has not been explored extensively. BH3-mimetics that inhibit specific BCL-2 anti-apoptotic proteins may hold encouraging treatment outcomes for cervical cancer management. Hence, the aim of this pilot study is to investigate the sensitivity of cervical cancer cell lines to combination of two BH3-mimetics namely ABT-263 which selectively inhibits BCL-2, BCL-XL and BCL-w and A-1210477, a selective MCL-1 inhibitor.We report that combination of A-1210477 and ABT-263 exhibited synergistic effects on all cervical cancer cell lines tested. Drug sensitization studies revealed that A-1210477 sensitised the cervical cancer cell lines SiHa and CaSki to ABT-263 by 11- and fivefold, respectively. Sensitization also occurred in the opposite direction whereby ABT-263 sensitised SiHa and CaSki to A-1210477 by eightfold. This report shows that combination of ABT-263 and A-1210477 could be a potential treatment strategy for cervical cancer. Extensive drug mechanistic studies and drug sensitivity studies in physiological models are necessary to unleash the prospect of this combination for cervical cancer therapy.The online version of this article (10.1186/s13104-018-3302-0) contains supplementary material, which is available to authorized users. Cervical cancer is the third leading cause of cancer death in females in less developed nations . The curThe BCL-2 family proteins, divided into pro- and anti-apoptotic proteins are the regulators of the intrinsic apoptosis pathway . The antNumbers of studies have evaluated the expression of the anti-apoptotic proteins at different stages of cervical cancer using immunoblot, immunohistochemistry (IHC) and oligonucleotide microarray profiling. Using IHC, BCL-2 and tumour suppressor protein p53 was detected to co-express in biopsies of cervical lesions but not in the normal cervical epithelium . EmployiABT-263 binds with high affinity to anti-apoptotic proteins BCL-2 and BCL-XL and with lower affinity to BCL-w , 11. ABTl-glutamine. C33A was maintained in DMEM medium supplemented with 10% (v/v) heat-inactivated FBS, 10 U/ml penicillin and 10 μg/ml streptomycin, 2 mM l-glutamine and 4.5 g/l d-Glucose, and CaSki was maintained in RPMI medium supplemented with 10% heat-inactivated FBS, 10 U/ml penicillin and 10 μg/ml streptomycin. The human foreskin fibroblasts were maintained in DMEM medium supplemented with 20% (v/v) FBS, 1 mM sodium pyruvate and 2 mM l-glutamine. All cell culture reagents were purchased from Gibco; Thermo Fisher Scientific, Waltham, MA, USA. Cells were incubated in 37 °C, 5% CO2. Unnecessary passaging of cell lines was avoided; experiments were conducted within two passages of reference stocks.SiHa was maintained in DMEM medium supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS), 10 U/ml penicillin and 10 μg/ml streptomycin, 1% (v/v) HEPES, 1 mM sodium pyruvate and 2 mM 2. After attachment, cells were treated to a concentration series [0–32 μM] of single agent ABT-263 and A-1210477 and as combination along the long plate axis for 72 h. Similar drug concentrations of ABT-263 and A-1210477 as previously described by [The 2D drug sensitivity assays were performed as described , 20. Celribed by , 21 were50 values of the combination with either single agent A-1210477 or ABT-263 were done by two-tailed paired T test using GraphPad Prism® version 7 software. The values of p-values are indicated as ***p ≤ 0.001 or ****p ≤ 0.0001.Comparisons of ICSynergistic drug interaction was determined using the median effect principle as previously described by Chou and Talalay , 23. TheDespite number of studies reporting on high expression of the anti-apoptotic proteins in cervical cancer tissues, targeting these proteins for therapy received very little attention. Here we conducted a preliminary study to investigate the sensitivity of three cervical cancer cell lines to BH3-mimetic ABT-263 which selectively inhibits BCL-2, BCL-XL and BCL-w and A-1210477, a selective MCL-1 inhibitor. The cells were tested to single agent activity of ABT-263 and A-1210477 and as combination. In order to determine that the drugs did have an effect on cell proliferation of normal cells, the drugs were tested on human foreskin fibroblasts (HFFs).50 11.13 ± 0.48 µM; CaSki IC50 3.4 ± 0.13 µM; C33A IC50 11.77 ± 0.11 µM—Fig. 50 20.23 ± 0.55 µM; CaSki IC50 6.7 ± 0.04 µM; C33A IC50 11.97 ± 0.22 µM—Fig. 50 3.07 ± 0.11 µM; CaSki IC50 1.6 ± 0.04 µM; C33A IC50 3.11 ± 0.06 µM—Fig. 50 of the drug combination in the HFFs was 8.1 ± 0.6 µM—Additional file 50 concentration obtained for the combination in the HFFs were higher compared to the cervical cancer cell lines (Fig. All three cervical cancer cell lines were resistant to single agent activity of ABT-263 (SiHa IC50 10.12 ± 0.34 µM; CaSki IC50 3.4 ± 0.13 µM – Fig. 50 2 ± 0.15 µM; CaSki IC50 0.3 ± 0.0013 µM—Fig. 50 19.86 ± 0.53 µM; CaSki IC50 7.6 ± 0.11 µM—Fig. 50: 2.58 ± 0.2 µM; CaSki IC50 0.9 ± 0.13 µM—Fig. Next, we investigated the sensitization effect of one drug by another in the SiHa and CaSki cells. We first tested the sensitization effect of ABT-263 by A-1210477 by adding a fix concentration of 4 μM of A-1210477 to increasing doses of ABT-263. SiHa and CaSki were resistant to single agent activity of ABT-263 (SiHa ICTaken together our data demonstrated that the drug combination synergistic anti-proliferative effects could be explained by the ability of both drugs to sensitize each other. Hence, ABT-263 and A-1210477 may be effective sensitizers at physiologically attainable doses.Neutralisation of MCL-1 is required for enhanced anti-cancer efficacy of ABT-263. Hence tumours that are normally unresponsive to ABT-263 may become amenable to treatment when combined with drugs which either repress MCL-1 or induce MCL-1 antagonist NOXA . In our In order to fully unravel the potential of combination of ABT-263 with its partner drug, we tested the sensitization of the cervical cancer cell lines to ABT-263 by A-1210477 and vice versa. In our hands, ABT-263 sensitized cervical cancer cell lines SiHa and CaSki to A-1210477 and vice versa demonstrating that both drugs can augment the activity of each other and restore the apoptotic potential in tumour cells. This was in agreement with other studies which reported that ABT-263 sensitized the effect of docetaxel in SKOV3 ovarian cancer xenograft model and erlotinib in the NCI-H1650 NSCLC xenograft model . AnotherA-1210477, similar to our findings, sensitized a number of cell lines from different cancer types namely BxPC-3 pancreas adenocarcinoma line, H23-lung carcinoma line, EJ-1 gastric carcinoma line and OPM-2 multiple myeloma line to ABT-263 in vitro . The senTaken together our data demonstrates that combination of ABT-263 and A-1210477 exhibited synergistic effects in all cervical cancer cell lines tested and both drugs have the ability to enhance the activity of each other at physiologically attainable concentrations. Combination of these drugs could be a potential therapy option to combat cervical cancer but further studies are necessary to fully unleash the prospect of this duo.Sensitivity of the cervical cancer cell lines to combination of ABT-263 and A-1210477 were performed in the 2D cell culture model. The 2D model is high-throughput and economical but it lacks the microenvironment that tumours encounter in vivo , 33. GivAdditional file 1. Anti-proliferative effects of ABT-263 and A-1210477 in human foreskin fibroblasts at 1:1 drug concentration ratios. This data shows the effect of the BH3 mimetic drug combination on cell viability of a non-cancerous cell line.Additional file 2. Synergistic drug effects of ABT-263 and A-1210477 in the SiHa and CaSki cervical cancer cell lines. This data shows the drug combination interaction effects in two cervical cancer cell lines.


To capture the molecular features and identify links with drug response, cancer cell lines remain the most highly characterized and high-throughput model system in use. An inherent question is to what extent cell lines recapitulate molecular and clinical diversity of Since the first large pharmacogenomics initiative of NCI-60 to analyze 60 cell lines , severalhttp://tcpaportal.org/tcpa/). Each cell line was profiled with an average of 230 high-quality antibodies that target total and phosphorylated proteins. The proteins are selected to provide a sparse unbiased coverage of all major functional and signaling pathways of relevance to cancer. A large subpopulation of these cell lines have been annotated at genomic and transcriptomic level and tested for drug sensitivity. A user-friendly web platform  was implemented to enable exploration of RPPA and related pharmacogenomics data. In addition to the rich information content, the most intriguing feature of MCLP is its integration with other large-scale multilayer data which will provide opportunities to advance our knowledge of protein features that define tumor subpopulations, protein network rewiring regulated by oncogenic lesions at DNA or RNA level, protein markers that predict therapeutic sensitivity, and identification of compounds likely to be active in select patient populations.In the MD Anderson Cell Lines Project (MCLP), normalized protein expression data of 706 cell lines (including 651 independent cell lines encompassing 19 lineages) were generated using reverse-phase protein arrays (RPPA), the same platform employed to characterize over 8,000 tumors in TCGA (Investigation of protein expression pattern in MCLP and TCGA  both revIntegrated analysis of proteomic and transcriptomic data from cell lines revealed moderate quantitative correlation and concordance in co-expression networks, which reinforced the limitation of using transcriptional profiles as surrogates for protein function. The combined analysis of both information levels is better able to capture compensatory feedback loops and posttranslational regulation, thus allows more accurate modeling of signaling and pathway activity. Similarly, mutations in cancer driver genes, as indicators of altered pathways, are frequently used to dictate treatments. However, the rewired signaling pathways may elicit indirect, sometimes unexpected effects that contribute to ultimate resistance and/or provides combinatorial therapeutic opportunities. These mutation-protein associations are now readily investigated in this large collection of highly characterized cell lines. Our analysis revealed that cell lines demonstrate the functional consequences of frequently mutated pathways like PI3K and p53 signaling in concordance with tumors.Identification of markers predictive of clinical benefit is a core goal for drug screening. A community effort  analyzinIn MCLP study, we explored therapeutic sensitivity of cell lines expressing protein markers of EMT pathways. Surprisingly a strong EMT signature was observed in cell lines from nearly all lineages assessed in MCLP. As expected, EMT was associated with resistance to the majority of therapeutic modalities. However, cells with a strong EMT signature demonstrated marked sensitivity to HMGCR targeting compounds and moderate sensitivity to a subset of drugs targeting the RAS/MAPK suggesting potential treatment options for this hard to treat population.The RPPA data and the interactive web portal collectively represent opportunities to explore tumor protein features in cancer cell lines and to capitalize on existing pharmacogenomics data to facilitate drug implementation.


Although chemo-immunotherapy has led to an improved overall survival for most B-cell lymphoma types, relapsed and refractory disease remains a challenge. The malaria drug artesunate has previously been identified as a growth suppressor in some cancer types and was tested as a new treatment option in B-cell lymphoma.We included artesunate in a cancer sensitivity drug screen in B lymphoma cell lines. The preclinical properties of artesunate was tested as single agent in vitro in 18 B-cell lymphoma cell lines representing different histologies and in vivo in an aggressive B-cell lymphoma xenograft model, using NSG mice. Artesunate-treated B lymphoma cell lines were analyzed by functional assays, gene expression profiling, and protein expression to identify the mechanism of action.. Artesunate induced potent growth suppression in most B lymphoma cells with an IC50 comparable to concentrations measured in serum from artesunate-treated malaria patients, while leaving normal B-cells unaffected. Artesunate markedly inhibited highly aggressive tumor growth in a xenograft model. Gene expression analysis identified endoplasmic reticulum (ER) stress and the unfolded protein response as the most affected pathways and artesunate-induced expression of the ER stress markers ATF-4 and DDIT3 was specifically upregulated in malignant B-cells, but not in normal B-cells. In addition, artesunate significantly suppressed the overall cell metabolism, affecting both respiration and glycolysis.Drug screening identified artesunate as a highly potent anti-lymphoma drugArtesunate demonstrated potent apoptosis-inducing effects across a broad range of B-cell lymphoma cell lines in vitro, and a prominent anti-lymphoma activity in vivo, suggesting it to be a relevant drug for treatment of B-cell lymphoma.The online version of this article (10.1186/s13045-018-0561-0) contains supplementary material, which is available to authorized users. More than 60 different B-cell lymphoma types have been described, with different clinical courses and outcomes . DiffuseArtemisia annua, and is the first-line treatment of severe malaria [Artesunate is a semisynthetic analogue of artemisinin, extracted from the sweet wormwood plant,  malaria . Artemis malaria , 6, alth malaria , 8. Few  malaria , 10. The malaria  and enabB lymphoma cell lines are representative models with > 80% match with the corresponding primary cancer cells , 13 , after informed consent and approval from regional authorities (REK S-03280). B-cells were isolated using anti-CD19 Dynabeads , followed by the corresponding DETACHaBEADs [Cell lines representing the most common B-cell lymphoma subtypes are the following: Burkitt lymphoma: BL-41, Raji, Ramos, Rec-1 (Leibniz-Institut-Deutche Sammlung von Mikroorganismen und Zellkulturen (DSMZ)) and Namalwa (gift from J. Delabie); Germinal Center B-like (GCB) DLBCL: ULA, SU-DHL-6, Oci-Ly-18 (DSMZ), Oci-Ly-2, SU-DHL-4 (gift from L. Staudt); activated B-cell like (ABC) DLBCL: U-2932 (DSMZ); DLBCL-double hit: Will-2 (DSMZ); mantle cell lymphoma: Mino, JeKo-1, Granta-519 (DSMZ); FL: Ros-50 (DSMZ), K-422 (gift from J. Delabie); immunoblastic B-cell lymphoma: DoHH-2 (DSMZ). K-422, Oci-Ly-2, Namalwa and Ros-50 were authenticated by STR-profile in 2011, BL-41, SU-DHL-4, SU-DHL-6, Ramos, Raji, Granta, WILL-2, Rec-1. Mino and DoHH-2 were DNA fingerprinted . The cell lines obtained as gifts were second generation passages. For routine use of the cell lines, second generation splits were thawed and the experiments were performed in the time frame of 1 week after thawing and to maximum 20 passages. Mycoplasma testing is always performed after 4 weeks for all cultures using PCR-based methods (Venor GeM Mycoplasma Detection Kit), and always prior to injection of cells into mice. Cell lines were cultured in RPMI-1640 supplemented with 10% fetal calf serum (FCS) or 10% human serum , penicillin, and streptomycin (complete media) and maintained at 37 °C in 5% COCHaBEADs . B-cellsThe gene encoding for luciferase-TropC-GFP  was subcwww.cytobank.org) [Cells were grown in 96-well plates  for 72 h with or without artesunate (0.125–8 μM) and small molecular inhibitors  . DL-buthlimma package [p value (FDR) of less than 0.01. The probes were collapsed according to gene symbol, using the annotation file for the Illumina’s HumanHT-12 v4 Expression BeadChip platform. When several probes mapped to the same gene, the probe with lowest log fold change was selected. The pathways and networks most enriched for the differential expressed genes were identified by Ingenuity Pathway Analysis (IPA) software (Qiagen) with default settings. Microarray data is available at NCBI’s Gene Expression Omnibus with accession number GSE94553 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE94553).Total RNA was isolated using MiRNeasy  after 4 and 12 h treatment with artesunate (5 μM). The microarray analyses were performed on Illumina’s HumanHT-12 v4 Expression BeadChip platform . The differential gene expression analysis was done using the  package  in R (veCells were lysed and processed for SDS-PAGE . Miniproscid Il2rgtm1Wjl/SzJ (NSG) mice were bred in-house. Pilot experiments were performed with three mice in each group. Based on the results, n = 10 for treatment and control group was chosen. The mice (6–10 weeks old) were injected subcutaneously with 2 × 106 BL-41 cells expressing firefly luciferase (BL-41-luc). Tumor take was measured by IVIS at day 4 before mice were divided into control and treatment groups. The mice were divided according to tumor size within each cage for either treatment or control group to have non-biased, comparable groups. Artesunate was dissolved in EtOH/DMSO (1:1) to 370 mg/ml and diluted 1:10 in 5% Na3CO3 before injections. Mice were injected daily from day 4 with 200 mg/kg artesunate intraperitoneally or control (5% EtOH and 5% DMSO in Na3CO3). Treatment was given for 12 days, then 2 days off, followed by treatment every day until day 19 . Tumor growth was monitored by bioluminescent imaging (IVIS spectrum in vivo imaging system) at regular intervals. Mice were injected intraperitoneally with 150 mg/kg d-luciferin  and imaged after 10 min. Inhalation anesthetic sevofluran (Baxter) supplemented with O2 and N2O was used during imaging. Caliper measurement was used to determine if the tumor size had reached maximum 2 cm in one direction or 2 cm3 that was the limit for euthanasia.The care and handling of animals for the present study were in conformity with the Norwegian Food Safety Authority in compliance with the European Convention of the Protection of Vertebrates Used for Scientific Purposes (Project ID 7729). NOD.Cg-Prkdc5 cells/well in DMEM XF unbuffered assay media, supplemented with 2 mM sodium pyruvate, 10 mM glucose, 2 mM l-glutamine and adjusted to physiological pH (7.6) and incubated in the absence of CO2 for 1 h prior to the seahorse measurements . Initially, the cell basal respiration was measured for all groups while after injection of oligomycin, a potent F1F0 ATPase inhibitor (1 μM), the OCR dropped by the amount required for ATP production by respiration. Subsequent addition of 1 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazon (FCCP) uncoupled the mitochondrial electron transport from ATP synthesis and hence revealed the maximal respiration capacity in each case. Finally, addition of 1 μM antimycin A and rotenone completely inhibited the electron transport and hence respiration. Initially, the basal glycolytic rates of the treatment groups were measured while upon addition of oligomycin to the cells, being unable to produce ATP by respiration switched to their highest glycolytic capacity to compensate for their ATP requirements.A Seahorse Extracellular Flux (XF96e) Analyzer  was used to measure the oxygen consumption rate (OCR) which relates to mitochondrial respiration and the extracellular acidification rate (ECAR) reflecting the cellular glycolytic activity of live intact B-cell lymphoma cells, in real time with and without artesunate pretreatment . Prior to the assay, BL-41, SU-DHL-6, and Mino cells were attached onto Cell-Tak  coated 96-well XF-PS plates at a density of 1 × 10t test and two-tailed heteroscedastic Student’s t test for Seahorse assay. One-sided t test was used for animal studies, in addition to log-rank test for the Kaplan-Meier plots. GraphPad Prism and Igor Pro were used for calculations. Differences were considered to be statistically significant if P < .05.Statistical significance was determined by two-tailed unpaired Student’s 50 values from 0.189 to 3.72 μM, and a high sensitivity to artesunate (IC50 < 1 μM) was observed in 11 out of 18 cell lines tested  or left untreated . Mice with varying tumor loads were evenly distributed among the groups . In comparison, the first artesunate-treated mouse was euthanized due to its tumor size 6 days later. All remaining mice were monitored after end of treatment (day 19), and a delayed tumor growth was observed in artesunate-treated mice. Survival analysis showed that the median time to reach maximum tumor size criteria was 17.5 versus 30.5 days for control and artesunate-treated mice, respectively . Other significant pathways identified by IPA were also related to stress response, including endoplasmic reticulum (ER) stress, tRNA charging, amino acid biosynthesis, and the protein ubiquitination pathway . Across the cell lines, 131 genes were commonly differentially expressed after 12 h and these genes had a very similar expression pattern Fig. . IngenuiP = 1.5 × 10−7) (data not shown). However, when all three cell lines were analyzed together, MYC was a central molecule in the top network  . We therP < .05). The broad effects of artesunate on metabolism are likely contributing to its potent anti-lymphoma activity in vivo.Given the well-established close interaction between ER and mitochondria, stress signals induced in the ER may negatively influence a variety of mitochondrial metabolic functions –26. It hIn this study, we tested small molecule inhibitors for potential tumor cell suppressive effects in B-cell lymphoma cell lines and compared with the anti-malaria drug artesunate. The screen identified artesunate as an attractive novel drug with potent activity across different cell lines, representative for various B-cell lymphoma histologies. Artesunate selectively and potently induced apoptosis in the B lymphoma cell lines but had minimal effect in normal B-cells. Of note, artesunate also showed potent anti-tumor effect in a highly aggressive lymphoma xenograft model.50 values for artesunate varying from 0.19 to 3.72 μM in B lymphoma cell lines. The strong apoptosis-inducing effect of artesunate at clinical relevant concentrations of artesunate is therefore promising.The standard treatment of malaria is artemisinin based combinatorial treatment for 3 days , and theMYC in BL-41, SU-DHL-4, and Oci-Ly-2, respectively). MYC was identified as a central molecule in the network analysis with all three cell lines combined, suggesting MYC involvement, and demonstrated that artesunate had potent effects independent of MYC translocation and mutational status. Furthermore, artesunate also potently induced apoptosis in WILL-2 and Oci-Ly-18 cells, representing “double hit lymphoma,” having aberrant overexpression of MYC and BCL2, and also in U2932, with a subclone with “double hit” aberrations [Artesunate has earlier been shown to overcome bortezomib resistance in myeloma cells in vitro  and to hrrations . This isrrations .The UPR was identified as the most deregulated pathway in response to artesunate. Additional top pathways activated by artesunate included tRNA charging, protein ubiquitination and amino acid biosynthesis, all connected to changes caused by UPR and ER stress , 38. ThiIn general, monotherapy in cancer treatment is unlikely to produce deep and lasting remission . TherefoOur results indicate that artesunate has potent growth suppressive effects restricted to malignant B-cells that warrant clinical testing in B-cell lymphoma patients.Additional file 1:Table S1. Overview of the additional drugs used in the first line screen. (DOCX 36 kb)Additional file 2:Figure S1. Artesunate induces apoptosis in B-cell lymphoma cell lines. (A) Dose response curves for the four most responsive cell lines and healthy CD19+ B-cells from Fig. n = 3-5, 0.125-8 μM artesunate, 72 h). (B) Propidium iodide (PI) staining detects a high level of dead cells in artesunate-exposed B-cell lymphoma cell lines  compared to healthy B-cells (not significant (n.s)). Cells were treated with artesunate  and stained with PI. The side scatter (SSC)- PI plot shows gating of the PI positive cells in untreated and artesunate treated BL-41 cells. (C) Artesunate induces apoptosis in B-cell lymphoma cell lines (P < 0.04), but not in healthy B-cells (n.s). Staining for active caspase 3 in lymphoma cell lines was done after artesunate treatment , fixation and permeabilization (PFA/methanol). The plots show example of gating for the positive active caspase 3 populations in both untreated and artesunate treated BL-41 cells. The cells are first gated on single cells, then living cells in forward/side scatter. (EPS 1906 kb)Additional file 3:Figure S2. No prominent cell cycle arrest was detected in B-cell lymphoma cell lines exposed to artesunate. Cells were incubated with artesunate (Art) , fixed and permabilized (PFA/MeOH), and stored at − 20 °C over night. Near IR-dye (Life technology) was used as live/dead stain. Cell cycle analysis was performed using Hoechst 33,258 (2 μg/ml) incubated for at least 15 min on ice. An LSRII with UV laser was used for measurement and Dean-Jett-Fox model in Flow Jo v 7 for data analysis. A stack plot of (n = 3) cell cycle phases is shown in three different cell lines treated with artesunate vs control. T-test with correction for multiple testing with Holm-Sidak correction a statistical difference (art vs control) was only found in Mino cells in G1/S phase (P = 0.001) . (EPS 1049 kb)Additional file 4:Figure S3. The tumor load is evenly distributed among the treatment and control groups. (A) Tumor growth was monitored with IVIS luminescence measurements . Image was taken at day 0 when treatment was started (day 4 after injection of BL-41-luc). (B) Graphical distribution of luminescence measurements at treatment start. (C) Artesunate treatment does not affect the weight of the mice. An increased weight was observed for both groups during the treatment. Shown is weight (g) after treatment start (n = 10 for each group). Weight at start was 16.0-22.8 g and the mice were 6-10 weeks old. (EPS 3428 kb)Additional file 5:Table S2. The top five pathways from the ingenuity pathway analysis (IPA) show UPR as the top regulated pathway in all three cell lines with corresponding p-value and percentage overlap of genes. (DOCX 232 kb)Additional file 6:Figure S4. Top network for all three cell lines in IPA. These networks show interactions between proteins that have an upregulation (red) or downregulation (green) in gene expression in the three cell lines stimulated with artesunate (5 μM) for 12 h compared to control (untreated sample). (A) BL-41, (B) Oci-Ly-2 and (C) SU-DHL-4. (EPS 15844 kb)Additional file 7:Figure S5. IPA upstream regulators analysis predicts MYC to be inhibited. (A) In the top list of the upstream regulators analyzed by IPA, ATF4 and TRIB3 are among the top genes. MYC on the other hand is listed as number ten. The activation z-score predicts if a gene is an upstream activator (red) or an upstream inhibitor (blue). This means that the genes are predicted to regulate other genes, which are shown in B for MYC. (B) Figure shows upstream regulated network for MYC in BL-41, Oci-Ly-2 and SU-DHL-4 merged and show an interaction map between MYC and other regulated genes in the arrays. The dark blue color of MYC means IPA predicts inhibiton of MYC with “more confidence”. The goal of the Regulator Effects analysis in IPA is to provide insight into the causes and effects of differentially expressed genes or proteins in a dataset, and how they might cause differences in downstream outcomes. (EPS 1096 kb)Additional file 8:Figure S6. BSO treatment decreases the levels of GSH and attenuates artesunate in lymphoma cell lines. (A) GSH-Glo glutathione assay was used to measure GSH levels after treatment with BSO  in BL-41, SU-DHL-6 and normal CD19+ B-cells with or without IL21. (B) BSO does not decrease cell viability in lymphoma cell lines after 24 h treatment (50 μM), a small decrease in cell viability was seen in normal B-cells (n.s). (C) Cells were treated with BSO (50 μM) for 24 h before co-treatment with increasing concentration of artesunate for 24 h. Pre-treatment with BSO induced an increased sensitivity to artesunate in malignant cells, but not in normal B-cells (n = 3 (cell lines), n = 2 CD19+ cells; two different donors). (EPS 1102 kb)


XylT) and the α‐fucosyltransferase (FucT) genes, using CRISPR/Cas9 genome editing, in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. In total, we knocked out 14 loci. The knocked‐out lines were stable, viable and exhibited a typical BY2 growing rate. Glycan analysis of the endogenous proteins of these lines exhibited N‐linked glycans lacking β‐xylose and/or α‐fucose. The knocked‐out lines were further transformed successfully with recombinant DNaseI. The expression level and the activity of the recombinant protein were similar to that of the protein produced in the wild‐type BY2 cells. The recombinant DNaseI was shown to be totally free from any xylose and/or fucose residues. The glyco‐engineered BY2 lines provide a valuable platform for producing potent biopharmaceutical products. Furthermore, these results demonstrate the power of the CRISPR/Cas9 technology for multiplex gene editing in BY2 cells.Plant‐produced glycoproteins contain N‐linked glycans with plant‐specific residues of β‐xylose and core α‐fucose, which do not exist in mammalian‐derived proteins. Although our experience with two enzymes that are used for enzyme replacement therapy does not indicate that the plant sugar residues have deleterious effects, we made a conscious decision to eliminate these moieties from plant‐expressed proteins. We knocked out the β‐xylosyltranferase ( Bright yellow 2 (BY2) cell line is among the most commonly used cells for this purpose  and β‐xylosyltransferase (XylT) ‐D‐mannose 4,6‐dehydratase enzyme, which is associated with GDP‐L‐fucose biosynthesis in Nicotiana benthamiana (N. benthamiana) plants. This resulted in N‐glycans with decreased α‐fucose residues ‐GnTI (N‐acetylglucosaminyltransferase I) antisense in potato (Solanum tuberosum) and in tobacco (N. tabacum) showed no significant changes in the total N‐glycan profiling versus the wild‐type plants ‐xylosyltransferase (XylT) and α‐fucosyltransferase (FucT) genes using targeted genome editing. Targeted genome editing using zinc finger nucleases —are known to be involved in the guiding process. The crRNA guides the Cas9 endonuclease to the specific DNA target, while the actual binding of the enzyme to the DNA is mediated by the tracrRNA , which identify and locate the targeted DNA sequence, and an associate DNA endonuclease (Cas9), which execute the sequence‐specific cleavage (Jinek N. tabacum genome is known to contain two XylT genes (Ntab‐XylT) and five FucT genes ‐xylose and α‐fucose plant‐specific glycans in the Protalix's BY2 cell system, enabling the production of recombinant glycoproteins lacking these sugar moieties.XylT‐A and Ntab‐XylT‐B  selectable marker under an internal ribosome entry site (IRES) sequence, followed by a human‐optimized Cas9 , a total of 250 individual lines were isolated and screened sequentially for the absence of fucose and xylose. Screening for fucose‐free lines was conducted by using an ELISA test. Total soluble protein from the putative fucose‐free lines (about 30% of the screened lines) was separated by SDS‐PAGE followed by a Western blot analysis using anti‐xylose. Twenty five  lines were assumed to be knocked out for both XylT and FucT.In the third transformation, using the phCas9‐XylT‐FucT vector Figure c and aimWestern blots of the total soluble protein extracted from three selected glyco‐engineered cell lines and the nontransgenic BY2 cells, using anti‐xylose, anti‐fucose or anti‐HRP antibodies, are presented in Figures XylT knocked‐out cell line (ΔXT), in a FucT knocked‐out cell line (ΔFT) and in a double knocked‐out cell line (ΔXFT).The mutations that were generated by the Cas9 were characterized in three different mutated cell lines, that is in a For each of the three cell lines, a PCR was performed using primers flanking the Cas9 target sites Table . The obtXylT‐A and a 1‐bp insertion and a 13‐bp deletion in XylT‐B  was compared with the glycans found in the wild‐type BY2 control cells. The glycan pools, separated on a normal phase UPLC system coupled with a fluorescence detector, showed a clear shift to shorter retention times [smaller glucose unit (GU) values] in the three mutated cell lines . In addition, a variety of high mannose glycans (Man6‐Man9) can be found. All glycans did not contain xylose or fucose.The prevalence of xylose, fucose and xylose‐/fucose‐containing glycans among the total glycans derived from each of the tested samples is summarized in Table WT) all contained xylose and fucose  contained either no xylose, no fucose or neither xylose nor fucose  or paucimannose, substituted with either one or two N‐acetylglycosamine sugars on the terminal mannose . The DNaseI glycan did not contain any high mannose glycans (Man6‐Man9) as expected from a secreted protein.To demonstrate that the knocked‐out cell lines can be used to produce recombinant proteins that do not contain plant‐specific sugar moieties, the glyco‐engineered cells were transformed with human recombinant DNaseI Figure  and compBY2 had two major peaks  and a monoanntenary core‐fucosylated and xylosylated peak . A third peak (4%) was identified as a paucimannose structure substituted with xylose and fucose (MMXF3). In the DNaseIΔXT, the main peak (92%) was assigned as a bianntenary core‐fucosylated glycan (GnGnF3) and the minor peak (8%) as a monoanntenary core‐fucosylated glycan . In the DNaseIΔFT, the main peak (56%) was assigned as a bianntenary glycan with xylose (GnGnX) and the minor peaks  as a monoanntenary glycan containing xylose (GnMX) or a paucimannose structure substituted with a bisecting xylose (MMX), respectively . The DNaseIΔXFT main peak (67%) was assigned as a nonfucosylated/nonxylosylated bianntenary glycan (GnGn), and its minor peaks (30% and 3%) were identified as a nonfucosylated/nonxylosylated monoanntenary glycan (GnM) or a paucimannose structure .The DNaseIs Figure  top—a peet al., Plants and plant cell suspensions in particular are considered as a promising platform for the production of biopharmaceuticals proteins ‐xylose and α‐fucose in glycoprotein is conserved in plants, the absence of an apparent difference between the WT and the knockout cell lines should be further studied. It might be that fucosylation and xylosylation of plant glycoproteins play a role under certain stress conditions, or during development in planta. In this respect, we show here, for the first time, that the presence of xylose or fucose sugars in glycoproteins is not vital for the growth of BY2 cells in culture.Aiming at eliminating the plant‐specific glycans β‐xylose and α‐fucose from recombinant glycoproteins that are produced in We were then able to utilize the new cell lines to produce a specific biotherapeutic protein. A subsequent transformation of the mutated lines with recombinant DNaseI showed that the biotherapeutic did not contain the β‐xylose and/or the α‐fucose in its glycans. This was used as a model to show that any recombinant glycoprotein can be produced in Protalix's glyco‐engineered BY2 cells.N. tabacum cv. BY2 cells to the list of plant systems that were shown to be amenable to targeted DNA modifications by applying the CRISPR/Cas9 technology. Furthermore, by simultaneously addressing and mutating seven genes, which are involved in the fucosylation and xylosylation of plant glycoproteins, and using one vector, it was demonstrated that the CRISPR/Cas9 technology can be efficiently used for multiplex genome editing in the BY2 cells. Note that in order to achieve loss of function, we had to produce bi‐allelic mutations in seven genes; therefore, a total of 14 genetic loci were knocked out. Earlier studies reported on the knockout of multiple loci in polyploid plants . Using the fucose antibodies, a direct ELISA was developed to enable a high‐throughput screening. Lines that were assumed to be knocked out for the XylT and FucT genes (Table Genomic DNA was extracted using the DNeasy plant mini kit (Qiagen). The DNA was amplified by PCR using specific primers for es Table . The PCRΔXT, BY2ΔFT, BY2ΔXFT) and nontransformed BY2 cells were transformed with DNAseI Table S3 Primers used for identification and characterization of the mutations in the knocked‐out cell linesClick here for additional data file.


U6, rRNA5s, SNORD44, SNORD24 and hsa-miR-24c-3p) using RedFinder algorisms to perform a stability expression analysis in i) normal colon cells, ii) colon and breast cancer cell lines and iii) cancer stem-like cell subpopulations. We identified SNORD44 as a suitable housekeeping gene for qPCR analysis comparing normal and cancer cells. However, this small nucleolar RNA was not a useful normalizer for cancer stem-like cell subpopulations versus subpopulations without stemness properties. In addition, we show for the first time that hsa-miR-24c-3p is the most stable normalizer for comparing these two subpopulations. Also, we have identified by bioinformatic and qPCR analysis, different miR expression patterns in colon cancer versus non tumour cells using the previously selected suitable normalizers. Our results emphasize the importance of select suitable normalizers to ensure the robustness and reliability of qPCR data for analyzing miR expression.Oncogenic microRNAs (miRs) have emerged as diagnostic biomarkers and novel molecular targets for anti-cancer drug therapies. Real-time quantitative PCR (qPCR) is one of the most powerful techniques for analyzing miRs; however, the use of unsuitable normalizers might bias the results. Tumour heterogeneity makes even more difficult the selection of an adequate endogenous normalizer control. Here, we have evaluated five potential referenced small RNAs ( MicroRNAs (miRs) are involved in the regulation of many physiological processes, including development, apoptosis and cell growth; as well as in pathological processes such as cancerogenesis267U6, rRNA5s, SNORD44 and SNORD24; however, their use has resulted in substantial discrepanciesTumour heterogeneity is characterized, in part, by different cell subpopulations including a small subset of cancer cells that act as initiating tumour cells or cancer stem cells (CSCs)67U6, rRNA5s, SNORD44 and SNORD24. We also used the hsa-miR-24c-3p since in some cases it has shown stability between several cell subpopulationsWe evaluated the expression pattern of the most commonly used small RNAs normalizers in HT-29 and HCT-116 colon cancer cell lines and in the CCD-18Co normal colon established cell line. We selected UniSp6 RNA spike from C. elegans. As shown in U6 was significantly overexpressed up to 500-fold (p < 0.01) in HCT-116 and up to 100-fold (p < 0.01) for HT-29 colon cancer cell lines in comparison with normal colon cell line. The rRNA5s also displayed increased expression levels in both HT-29 and HCT-116 colon cancer cell lines up to 2.3-fold (p < 0.01) and 1.48-fold (p < 0.01), respectively. Moreover, SNORD24 was significantly overexpressed in HT-29  and HCT-116  colon cancer cell lines in comparison with the CCD-18Co normal colon cell line. In contrast, the hsa-miR-24c-5p was significantly downregulated in both HT-29 and HCT-116 respect to the CCD-18Co normal colon cell line. Interestingly, SNORD44 did not show significant differences in colon cancer cell lines in comparison with epithelial colon normal cells  low adherent cells isolated by differential trypsinization, as we have previously described in Morata-Tarifa et al. 2016+ human colon cancer cells and MDA-MB-231 trypsin sensitive (TS) human breast cancer cells, with their corresponding ALDH− and trypsin resistant (TR) subpopulations with not stemness properties. The artificial UniSp6 RNA spike from C. elegands was used as control. Since SNORD24 showed a weak expression in these cell subpopulations, it was discarded as a potential housekeeping candidate.For the selection of small RNAs that could be used as normalizers in qPCR analysis of miR expression for cancer stem-like cell subpopulations, we analyzed two cell subpopulations with stemness properties: i) ALDHSNORD44 was analyzed since it was the one showing greater stability between tumour and non tumour cell lines , TargetScan 6.0 (www.targetscan.org) and Pictar (http://pictar.mdc-berlin.de/) to identify predicted miRs targeting mRNAs involved in self-renewal pathways  and other CSC related pathways as shown in SNORD44 as normalizer based on our previously described results. As shown in miR-142-3p. However, miR-590-5p and miR-15b-5p were overexpressed in HT-29 cells, while in HCT-116 did not display significant changes. In addition, a dramatic decrease in miR-199a-5p and miR-370 expression levels for the two tumour cell lines was observed when compared to the CCD18-Co non-tumour cell line. The analysis of selected miRs in cancer stem-like subpopulation using the hsa-miR-24c-3p normalizer was previously published in Morata-Tarifa C et al. 2016Once identified the most stable normalizer for miR expression analysis for each cell line or each CSC subpopulation, we decided to undertake a bioinformatic study for the identification of miRs targeting mRNAs involved in cancer pathways. We used miRanda-mirSVR  and HT-29 (ATCC HTB-38), the triple negative human breast cancer cell line MDA-MB-231 (ATCC HTB-26), and the normal human colon cell line CCD-18Co (ATCC CRL1459) were obtained from American Type Culture Collection (ATCC) and cultured according to the ATCC indications.et al. 2016et al. 2016Selection of cancer cells with CSC-like properties or with a more differentiated phenotype was performed using the differential trypsinization method as we previously described by Morata-Tarifa C ® kit assay  according to the manufacturer’s instructions. Analyses were performed in a FACS CANTO II (BD Biosciences) cytometer using the FACS DIVA software.Aldehyde dehydrogenase (ALDH) enzyme activity in viable cells was assayed using AldefluorPredictions were realized looking for miRs targeting mRNA of proteins that enhance or inhibit CSC-related pathways . miRs weUniSp6 RNA Spike-in control primer set is used to amplify UniSp6 RNA Spike-in template for the control of the cDNA synthesis step. The standard curve was constructed by 5-fold serial dilutions of cDNA.Total RNA, including miRs, was obtained from the different cell lines and subpopulations using the miRNeasy Mini Kit  following manufacturer´s instructions. Reverse transcription from miRs was performed using the miRCURY LNATM Synthesis kit II  following manufacturer’s protocol. qPCR was performed using miRCURY LNA TM EXILENT SYBR Green (Exiqon) in a CFX96 real-time PCR detection system (Bio-Rad). Each reaction was performed in triplicate and the comparative threshold cycle (Ct) method was used to calculate the amplification factor. The stability of candidate reference miRs was evaluated using RefFinder see , a user-All graphs present mean ± SEM from ≥ 3 assays. Student’s t-test was used for experiments with two groups. Comparisons of > 2 groups used one way analysis of variance (ANOVA) followed by Dunnett’s or Tukey’s post hoc analysis. Some experiments used two way analysis of variance followed by Tukey´s post hoc tests. P-values < 0.05 were considered statistically significant in all cases.How to cite this article: Morata-Tarifa, C. et al. Validation of suitable normalizers for miR expression patterns analysis covering tumour heterogeneity. Sci. Rep.7, 39782; doi: 10.1038/srep39782 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Oncogenic FLT3 kinase is a clinically validated target in acute myeloid leukemia (AML), and both multi-targeted and selective FLT3 inhibitors have been developed. Spleen tyrosine kinase (SYK) has been shown to be activated and increased in FLT3-ITD-positive AML patients, and has further been shown to be critical for transformation and maintenance of the leukemic clone in these patients. Further, over-expression of constitutively activated SYK causes resistance to highly selective FLT3 tyrosine kinase inhibitors (TKI). Up to now, the activity of the multi-targeted FLT3 inhibitor, midostaurin, against cells expressing activated SYK has not been explored in the context of leukemia, although SYK has been identified as a target of midostaurin in systemic mastocytosis. We compared the ability of midostaurin to inhibit activated SYK in mutant FLT3-positive AML cells with that of inhibitors displaying dual SYK/FLT3 inhibition, targeted SYK inhibition, and targeted FLT3 inhibition. Our findings suggest that dual FLT3/SYK inhibitors and FLT3-targeted drugs potently kill oncogenic FLT3-transformed cells, while SYK-targeted small molecule inhibition displays minimal activity. However, midostaurin and other dual FLT3/SYK inhibitors display superior anti-proliferative activity when compared to targeted FLT3 inhibitors, such as crenolanib and quizartinib, against cells co-expressing FLT3-ITD and constitutively activated SYK-TEL. Interestingly, additional SYK suppression potentiated the effects of dual FLT3/SYK inhibitors and targeted FLT3 inhibitors against FLT3-ITD-driven leukemia, both in the absence and presence of activated SYK. Taken together, our findings have important implications for the design of drug combination studies in mutant FLT3-positive patients and for the design of future generations of FLT3 inhibitors. FLT3 -3-dependent murine pro-B) cells engineered to express FLT3-ITD, SYK-TEL, TEL-SYK, FLT3-ITD+SYK-TEL, and FLT3-ITD+TEL-SYK  were proHuman AML-derived, FLT3-ITD-expressing MV4,11 cells were obtained from Dr. Anthony Letai. The human AML-derived, FLT3-ITD-expressing line, MOLM14 , was proThe human AML-derived, FLT3-ITD-expressing cell line, MOLM-13 , was engineered to express luciferase fused to neomycin phosphotransferase (pMMP-LucNeo) by transduction with a VSVG-pseudotyped retrovirus as previously described . Develop2 at 37°C, at a concentration of 2×105 to 5×105 in RPMI  with 10% fetal bovine serum (FBS) and supplemented with 2% L-glutamine and 1% penicillin/streptomycin. Parental Ba/F3 cells and Ba/F3-wt FLT3 cells were cultured in RPMI with 10% FBS and supplemented with 2% L-glutamine and 1% penicillin/streptomcyin, as well as 20% WEHI (as a source of IL-3).All cell lines used in this study were cultured with 5% COMycoplasma-free.Human cell lines were submitted for cell line authentication and were authenticated within 6 months of manuscript preparation through cell line short tandem repeat (STR) profiling . All cell lines tested matched >80% with lines listed in the ATCC or DSMZ Cell Line Bank STR. All cell lines were confirmed to be virus- and TM protease inhibitor cocktail, EDTA-free (100X) . Protein lysates were incubated on ice for 25 min, vortexed at 5 min intervals, and then centrifuged for 15 min at 12,000 X g. Supernatants were saved, and the Bio-Rad Protein Assay was used to determine protein yields . Equivalent amounts of protein were then loaded onto a gel for immunoblotting. For immunoprecipitation, cell lysates were incubated with antibody and protein A/G Sepharose overnight with rocking at 4°C. After the incubation, immune complexes were washed twice with lysis buffer, twice with 1X PBS, and were then resuspended in Laemmeli's sample buffer and boiled for 5 min. For immunoblotting and immunoprecipitation, samples were resolved on a sodium dodecyl sulfate (SDS)-10% polyacrylamide gel. Then proteins were electrophoretically transferred to a Protran nitrocellulose transfer and immobilization membrane . The membrane was then blocked overnight at 4°C with BSA or milk in 1X TBS  and then probed with antibody overnight at 4°C in 1X TBST buffer . After three washes with 1X TBST, membranes were incubated for 1 hr at 25°C with anti-mouse immunoglobulin (horseradish peroxidase-linked whole antibody from sheep) . The membrane was washed 5X in 1X TBST buffer, with 5 min intervals between buffer changes, and bound antibody was detected with enhanced luminol and oxidizing reagent as specified by the manufacturer .Protein lysate preparation, immunoblotting, and immunoprecipitation were carried out as previously described . BrieflyThe following antibodies were purchased from Cell Signaling Technology : total AKT  and total p44/42 MAPK (Erk1/2) (3A7)  were used at 1:1000. Anti-GAPDH (D16H-11) XP (R)  was used at 1:1000. Phospho-AKT (Ser 473) (D9E) XP(R)  was used at 1:1000. Phospho-p44/42 MAPK (T202/Y204)  was used at 1:1000. Phospho-S6 ribosomal protein (S235/236) (D57.2.2E) XP (R)  was used at 1:1000. Total S6 ribosomal protein (SG10)  was used at 1:1000. Phospho-STAT5 Tyr694  and total STAT5 (3H7)  were used at 1:1000. The Syk (D3Z1E) XP  was used at 1:1000.FLT3/Flk-2 (C-20) (sc-479) was purchased from Santa Cruz Biotechnology, Inc.,  and used at 1:1000 for immunoblotting. Anti-pTyr  was purchased from Upstate Biotechnology  and was used at 1:1000 in the presence of 4% BSA.The trypan blue exclusion assay has been previously described  and was Programmed cell death of inhibitor-treated cells was determined using the Annexin-V-Fluos Staining Kit , as previously described . BrieflyCell cycle analysis was performed as previously described . BrieflyFor measurement of caspase-3 and -7 activity, the Apo-ONE Homogenous Caspase-3/7 Assay kit was used . The assay was carried out according to manufacturer's instructions.50 or IC50 of each drug, combinations are studied where the concentrations are multiples, or fractions, of the ED/IC50. Statistical analysis is automatically part of the computations. Combination indices, values generated by the Calcusyn software, which are less than one indicate synergy, whereas values greater than one indicate antagonism. Calcusyn combination indices can be interpreted as follows: CI <0.1 indicate very strong synergism; values 0.1-0.3 indicate strong synergism; values 0.3-0.7 indicate synergism; values 0.7-0.85 indicate moderate synergism; values 0.85-0.90 indicate slight synergism; values 0.9-1.1 indicate nearly additive effects; values 1.10-1.20 indicate slight antagonism; values 1.20-1.45 indicate moderate antagonism; values 1.45-3.3 indicate antagonism; values 3.3-10 indicate strong antagonism; values >10 indicate very strong antagonism.For drug combination studies, cell viability was first determined using the Trypan Blue exclusion assay to quantify cells for cell seeding, and Cell Titer Glo was then implemented for proliferation studies. Single agents were added simultaneously at fixed ratios to cells. Cell viability was expressed as the function of growth affected (FA) drug-treated versus control cells; data were analyzed by Calcusyn software , which was utilized for synergy measurement and based on isobologram generation and the method of Chou-Talalay (1984) . This me


Differentiated PKM2 and GLS1 expression in both THC8307 and THC8307/Oxa cell lines was identified. In the THC8307 cell line, PKM2 and GLS1 can accelerate malignant behaviors, increase oxaliplatin-resistance, upregulate Pgp expression, and inhibit cell apoptosis. Contrastingly in the THC8307/Oxa cell line, knockdown of PKM2/GLS1 expression can restrain malignant behaviors, reestablish oxaliplatin-sensitivity, downregulate Pgp expression, and induce cell apoptosis. In xenograft, knockdown of PKM2/GLS1 expression can significantly inhibit tumor growth, reduce Pgp expression, and increase tumor apoptosis. Taken together, the present findings enriched our knowledge by demonstrating a significant association of PKM2 and GLS1 with oxaliplatin-resistance in CRC. We further propose that knockdown of PKM2/GLS1 expression may constitute a novel therapeutic strategy toward effective treatment for CRC.Clinical treatment for colorectal cancer (CRC) thus far encounters a huge challenge due to oxaliplatin-resistance. As crucial rate-limiting enzymes in aerobic glycolysis and glutaminolysis, pyruvate kinase M2 type (PKM2) and kidney-type glutaminase (GLS1) are proposed to carry important implications in colorectal carcinogenesis and drug-resistance. This study aimed to explore the possible association of oxaliplatin-resistance with aerobic glycolysis/glutaminolysis indexed by PKM2/GLS1 expression. PKM2 and GLS1 expression was quantified by polymerase chain reaction (PCR) and Western blot techniques in CRC cell lines. The abilities of cell formation, kinetics, migration, invasion, survival and apoptosis, as well as permeability glycoprotein (Pgp) expression were inspected before and after knocking-down PKM2/GLS1 expression. In addition, the influence of knocking-down PKM2/GLS1 expression was evaluated  Colorectal cancer (CRC) ranks as the third most common cancer around the world. Globally, over 1 million people are newly diagnosed to develop CRC per annum . CurrentIt is widely recognized that some alterations are necessitated for cancer cells to adapt in response to chemotherapeutic stress for survival . Also, tin vitro and in vivo were observed and tested, striving to elucidate the potential molecular mechanisms of oxaliplatin-resistance in CRC cells.To explore the possible association of oxaliplatin-resistance with aerobic glycolysis and glutamine metabolism, we sought to employ small interfering RNA (siRNA) to knockdown the expression of PKM and GLS1. Then, malignant behaviors, 50% inhibiting concentration (IC50), permeability glycoprotein (Pgp) expression, and cell apoptosis As indicated by qRT-PCR (quantitative reverse transcription - polymerase chain reaction) technique, PKM2 mRNA expression was 3.7 ± 0.53  and 2.56 ± 0.11 in CRC cell line (THC8307) and in oxaliplatin-resistant CRC cell line (THC8307/Oxa), respectively, as compared with that in the HCMEC cell line. PKM2 expression was significantly higher in the THC8307 cell line than in the THC8307/Oxa cell line, and it was also significantly higher in the THC8307/Oxa cell line than in the HCMEC cell line (both p < 0.05). Similarly, GLS1 mRNA expression was 3.34 ± 0.38 and 9.69 ± 0.17 in the THC8307 and THC8307/Oxa cell lines respectively, as compared with that in the HCMEC cell line, the differences being statistically significant (both p < 0.05) Figure . SubsequAfter siRNA transfection, PKM2/GLS1 expression was further confirmed in the THC8307 and THC8307/Oxa cell lines. As shown in Figure Colony formation, wound healing, Transwell test, MTS test and IC50 calculation were performed in both THC8307 and THC8307/Oxa cell lines. Before knocking-down PKM2/GLS1 expression, the THC8307/Oxa cell line exhibited the most significant cell formation ability Figure , 3B, wouAfter knocking-down PKM2/GLS1 expression in the THC8307 and THC8307/Oxa cell lines, the substantial inhibitory efficiencies of cell formation ability Figure  and 5B, Immunofluorescence assay confirmed that the THC8307/Oxa cell line exhibited more Pgp expression Figure , 6B. AftAnnexin V/PI assay revealed that the THC8307/Oxa cell line (apoptosis rate: 1.98 ± 0.28) had a more anti-apoptosis ability, as compared with the THC8307 cell line (apoptosis rate: 3.75 ± 0.12). After knocking-down PKM2/GLS1 expression in the THC8307 and THC8307/Oxa cell lines, apoptosis rate was significantly boosted in the esiPKM2+siGLS1 group Figure  and B2.th day ; NcsiRNA (100 μg/0.02 mL) plus oxaliplatin (5 mg/kg) (group II); siPKM2 (100 μg/0.02 mL) plus siGLS1 (100 μg/0.02 mL) (group III); siPKM2 (100 μg/0.02 mL) plus siGLS1 (100 μg/0.02 mL) plus oxaliplatin (5 mg/kg) (group IV). At the beginning, the tumors of 4 groups grew in a similar tendency, while tumor growth was retarded significantly in group IV at the 17y Figure . Consists Figure  and 8C.IHC analyses were used to quantify the expression of PKM2, GLS1 and Pgp in xenograft. As shown in Figure Tunel assay was employed to detect apoptosis rate in xenograft. As shown in Figure Cancer is increasingly recognized as a metabolic disease featured by energy imbalance . At the Our initial findings identified significantly elevated expression of PKM2 in the THC8307 cell line relative to the HCMEC cell line, at both mRNA and protein levels, which supported the notion that PKM2 might participate in the development of CRC. Further, we found that PKM2 expression was significantly higher in the THC8307 cell line than in the THC8307/Oxa cell line, the finding in agreement with that in the study by Martinez et al., who reported that PKM2 downregulation was involved in oxaliplatin-resistance in CRC , 29. In Considering the important role of PKM2 and GLS1 in CRC growth based on above evidence, we were inspired to investigate the influence of knocking-down PKM2/GLS1 expression in CRC cells. To fully address this issue, we have successfully constructed two siRNAs that were specifically targeted on PKM2 and GLS1, and found that both siRNAs could normally take effect without interference. After knocking-down PKM2/GLS1 expression, reduced IC50 was observed in both THC8307 and THC8307/Oxa cell lines, suggesting that PKM2/GLS1 downregulation could enhance the sensitivity for oxaliplatin and/or reverse drug-resistance in CRC cell lines. These observations agreed with the findings of previous studies that reported similar consequences of siPKM2 and siGLS1 in lung cancer and breast cancer cells , 33. TheAs an energy-dependent transport protein, Pgp is involved in multi-drug resistance in cancer, and it further results in chemotherapy failure . In the Anti-apoptosis represents another important mechanism for oxaliplatin-resistance . As revein vivo, and found that their knockdown exerted a significant tumor-inhibitory impact in xenograft. Correspondingly, Pgp expression was suspended, and apoptosis rate was elevated. There is strong agreement between in vivo and in vitro data, indicating the robustness of our findings.Finally, we examined the efficiency of knocking-down PKM2/GLS1 expression A major drawback of the present study is that only one cell type (THC8307) was employed to investigate the relationship between PKM2/GLS1 expression and oxaliplatin-resistance in CRC. Bearing this drawback in mind, we will design a protocol for following investigations more elaborately and make results more reliable.In summary, the present findings enriched our knowledge by demonstrating a significant association of two key rate-limiting enzymes, PKM2 and GLS1, with oxaliplatin-resistance in CRC. Moreover, differentiated expression of PKM2 and GLS1 in CRC cell lines can increase oxaliplatin-resistance by raising Pgp expression and inhibiting cell apoptosis, whereas in drug-resistance CRC cell lines, the knockdown of PKM2/GLS1 expression can restore oxaliplatin-sensitivity by degrading Pgp expression and inducing cell apoptosis. This present study deepens our understanding of molecular mechanisms underlying chemotherapy resistance of oxaliplatin in CRC cells by proposing that knockdown of PKM2/GLS1 expression may constitute a novel therapeutic strategy toward effective treatment for CRC in clinical practice.Highly differentiated CRC cell line (THC8307) and oxaliplatin-resistant CRC cell line (THC8307/Oxa) were purchased from the Saierbio Co., Ltd. . Normal human colon mucosa epithelial cell line (HCMEC) was kindly provided by Sun Yat-sen University Cancer Center. Oxaliplatin was purchased from the Sanofi-Avenitis . Anti-PKM2 was purchased from the Santa Cruz Co., Ltd. . The other antibodies were purchased from the Abcam . All study protocols followed standard guidelines, and were approved by the Human Research Ethics Committees of Sun Yat-sen University.Both qRT-PCR and WB techniques in two CRC cell lines, THC8307 and THC8307/Oxa, were performed to quantify the PKM2/GLS1 expression, as previously described , 38. TheThe GLS1/PKM2-siRNAs were chemically synthesized by the Sigma Chemical Co.,Ltd. . The sequences of siRNAs were as follows: for GLS1-siRNA1: 5′- GUU GAA AGA GUG UAU GGA UdT dT -3′ (forward) and 5′- AUC CAU ACA CUC UUU CAA CdT dT -3′ (reverse); for PKM2-siRNA2: 5′ -GAA UGA AUG UGG CUC GUC UdT dT -3′ (forward) and 5′- AGA CGA GCC ACA UUC AUU CdT dT -3′ (reverse). Then, the efficiency of siRNA transfection of PKM2/GLS1 in the THC8307 cell line was examined by WB technique. The NcsiRNA was used as the negative control.Colony formation, cell scratch, Transwell test, MTS test and IC50 calculation were performed in two CRC cell lines - THC8307 and THC8307/Oxa, before knocking-down PKM2/GLS1 expression. After its knockdown in the THC8307 cell line, all phenotype experiments were re-performed. All protocols were performed according to the methods described previously –41. The Pgp expression was detected by immunofluorescence assay before knocking-down PKM2/GLS1 expression. Both THC8307 and THC8307/Oxa cell lines were cultured in 6-well plates. After incubating and washing, the plate was blocked with 1% non-fat milk for 30 min, followed by administration of anti-Pgp antibody . Then, a horseradish peroxidase (HRP) antibody was added . After knocking-down PKM2/GLS1 expression in the THC8307 cell line, Pgp expression was re-tested by immunofluorescence assay. The protocols were performed according to the methods described previously . The HCMApoptosis rate of two CRC cell lines (THC8307 and THC8307/Oxa) was investigated by Annexin V/PI assay before knocking-down PKM2/GLS1 expression, as previously described . After k7 THC8307/Oxa cells per mL were washed twice with PBS, and were injected subcutaneously in a volume of 0.1 mL into the flank of mice. After inoculation, tumor-bearing mice were divided randomly into 4 treatment groups: THC8307/Oxa negative control (group I); NcsiRNA (100 μg/0.02 mL) plus oxaliplatin (5 mg/kg) (group II); siPKM2 (100 μg/0.02 mL) plus siGLS1 (100 μg/0.02 mL) (group III); siPKM2 (100 μg/0.02 mL) plus siGLS1 (100 μg/0.02 mL) plus oxaliplatin (5 mg/kg) (group IV). Animal experiments were conducted in accordance with the public health service policy, and they were approved by the Animal Care and Use Committees of Sun Yat-sen University. Mice bearing tumors were observed, and tumor size was measured once every 3 days using vernier caliper. At the end of the experiments (at the 28th day), all animals were killed, and the tumors from each animal were removed and prepared [Sixteen 4-week-old SCID mice with body weight of approximately 20 g were purchased from the Animal Center of Sun Yat-sen University, and they were raised under specific pathogen-free conditions. For tumor establishment, 2 × 10prepared .IHC analysis was performed as previously described to explore the expression of PKM2, GLS1 and Pgp . SimultaQuantitative data were described as means ± standard deviation. Two-group comparisons of quantitative data were completed by using the Student's t-test. Statistical significance was declared when the two-tailed p-value is 0.05 or less. Statistical analyses were accomplished with the SPSS Statistics Version 22.0 .


Cell migration is essential for development and tissue repair, but it also contributes to disease. Rho GTPases regulate cell migration, but a comprehensive analysis of how each Rho signalling component affects migration has not been carried out.Through an RNA interference screen, and using a prostate cancer cell line, we find that approximately 25% of Rho network components alter migration. Some genes enhance migration while others decrease basal and/or hepatocyte growth factor-stimulated migration. Surprisingly, we identify RhoH as a screen hit. RhoH expression is normally restricted to haematopoietic cells, but we find it is expressed in multiple epithelial cancer cell lines. High RhoH expression in samples from prostate cancer patients correlates with earlier relapse. RhoH depletion reduces cell speed and persistence and decreases migratory polarity. Rac1 activity normally localizes to the front of migrating cells at areas of dynamic membrane movement, but in RhoH-depleted cells active Rac1 is localised around the whole cell periphery and associated with membrane regions that are not extending or retracting. RhoH interacts with Rac1 and with several p21-activated kinases (PAKs), which are Rac effectors. Similar to RhoH depletion, PAK2 depletion increases cell spread area and reduces cell migration. In addition, RhoH depletion reduces lamellipodium extension induced by PAK2 overexpression.We describe a novel role for RhoH in prostate cancer cell migration. We propose that RhoH promotes cell migration by coupling Rac1 activity and PAK2 to membrane protrusion. Our results also suggest that RhoH expression levels correlate with prostate cancer progression.The online version of this article (10.1186/s12915-018-0489-4) contains supplementary material, which is available to authorized users. Cell migration is essential for the development of multicellular animals, as well as for immune responses and wound healing. Cell migration also contributes to the development of several human diseases, including cancer, heart disease and chronic inflammatory disorders –3. Many Rho GTPases are intracellular coordinators of cell migration signalling. Most Rho GTPases cycle between an active GTP-bound and an inactive GDP-bound form. They are activated by guanine nucleotide exchange factors (GEFs), which stimulate exchange of GDP for GTP, and inactivated by GTPase-activating proteins (GAPs), which stimulate their intrinsic GTPase activity, hydrolysing GTP to GDP . Most RhSeveral groups have used RNA interference (RNAi) screens to identify regulators of cell morphology and/or cell migration. Targeted screens of cytoskeletal regulators as well as whole genome screens have identified new genes involved in cell migration –18.Here we have carried out a focussed RNAi screen to identify which Rho GTPases and their interacting partners regulate cancer cell migration. Unexpectedly, we identified RhoH as a key regulator of cancer cell migration. RhoH is widely assumed to be expressed only in haematopoietic cells based on analysis of mouse tissues . We showTo investigate which Rho GTPase network components affect cell migration, we used a small interfering RNA (siRNA) library containing pools of four siRNAs targeting each of 202 genes encoding Rho GTPases, RhoGEFs, RhoGAPs and Rho targets, together with four control siRNA pools  to allow rapid quantification of relative cell number in the wound area. Images were treated with a flatten background filter and then thresholded so that differences in CFSE uptake between individual cells did not affect the results Fig. . This waCdc42 depletion strongly reduced cell migration Fig. . We compThe siRNA library screen was repeated three times. To identify if any siRNA pools affected cell viability, PC3 cells were plated onto 96-well plates 24 h after transfection, and the fluorescence was measured after 24 and 48 h to monitor cell viability Fig. . Only a Screen results were analysed by calculating the migration rate for each individual siRNA pool compared to the mean of all the wells in the screen Fig. . A Z-scoIt is interesting that siRNA pools for 6 of the 20 (30%) Rho GTPase family members altered cell migration Fig. . All 20 In total, siRNA pools for 20 RhoGEFs affected cell migration, of the 70 included in the library (29%). All of the 9 RhoGEF siRNA pools that reduced basal cell migration are reported to be GEFs for Rac and/or Cdc42, consistent with roles of Rac and Cdc42 in lamellipodium-driven migration . For RhoOf the 57 Rho effectors in our library, 15 are hits (26%)  Fig. . TogetheRhoH is expressed in PC3 cells, we investigated whether other cancer cell lines used in our laboratory also expressed RhoH. RNA analysis by polymerase chain reaction (PCR) detected RhoH expression in PC3 cells and MCF7 breast cancer cells but not DU145 prostate cancer cells or MDA-MB-231 breast cancer cells  database (http://www.broadinstitute.org/ccle). As expected, the highest RhoH expression levels were found in T- and B-cell-derived cancers including lymphoma and leukemia cell lines. RhoH was also expressed in a variety of epithelial cancer cell lines, including prostate, breast, colorectal and lung. The 8 prostate cancer cell lines and 58 breast cancer cell lines in the CCLE database expressed RhoH mRNA at a wide range of levels. Based on this analysis, we tested for RhoH expression in a selection of cancer cell lines, including those with high and low expression based on our CCLE analysis. Quantitative real-time PCR (qPCR) analysis showed that, of the cancer cell lines analysed, RhoH mRNA levels were highest in T47D breast cancer cells followed by LNCaP and PC3 prostate cancer cells, whereas RhoH mRNA was very low or not detected in a variety of other cancer cell lines  /[Perimeter], with a value of 1.0 indicating a perfect circle; as the value approaches 0.0, it indicates an increasingly elongated shape. Shape factor = 4πA/P2; a value near 0 indicates a flattened object, whereas a value of 1.0 indicates a perfect circle. Elongation factor = 1/Shape factor.4) were plated on 6-well tissue culture dishes that were pre-coated for 1 h with 100 μg/ml Matrigel (BD Biosciences). After 16 to 24 h, the cells were transfected with 100 nM siRNAs. Cells were counted at 48 h, 72 h or 96 h after transfection using a haemocytometer.PC3 cells .The MSKCC prostate cancer mRNA expression dataset and descriptive statistics of associated clinical data were obtained from the cBio Cancer Genomics Portal . RhoH mRt test (for comparing two conditions) or by one-way analysis of variance (ANOVA) with multiple conditions, or a Wald test .All data are presented as mean plus SEM. The statistical analysis was performed using GraphPad Prism software. The statistical significance analysis was calculated using an unpaired two-tailed Using an RNAi screen, we show that multiple Rho GTPases and their interacting partners, including GEFs, GAPs and effectors, regulate cell migration in PC3 prostate cancer cells. One of the screen hits is RhoH, which was assumed to be expressed only in haematopoietic cells, but we show is expressed in a panel of epithelial cancer cell lines. RhoH promotes cell migration and chemotaxis of PC3 cells. RhoH co-localises with and associates with Rac1 and PAK2, a Rac1 effector and a hit in our RNAi screen, and promotes coupling of Rac1 activity to membrane protrusion events as well as PAK2 localisation to lamellipodia. We propose that RhoH stimulates Rac1- and PAK2-driven membrane protrusion and hence enhances cell migration.Additional file 1:Table S1. List of gene targets in siRNA library. Genes are divided into controls, Rho GTPases, other GTPases, RhoGEFs, RhoGAPs, effectors and others. Gene names and alternative gene names, Entrez Gene GeneIDs, RefSeq accession numbers, siRNA sequences and effects of each gene on migration and cell viability are shown as Z-scores. (XLSX 49 kb)