The Experts below are selected from a list of 1953 Experts worldwide ranked by ideXlab platform
Franzgeorg Hanisch - One of the best experts on this subject based on the ideXlab platform.
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recombinant muc1 probe authentically reflects cell specific o glycosylation profiles of endogenous breast cancer mucin high density and prevalent core 2 based glycosylation
Journal of Biological Chemistry, 2002Co-Authors: Stefan Muller, Franzgeorg HanischAbstract:Abstract Knowledge about the O-linked glycan chains of tumor-associated MUC1 is primarily based on enzymatic and immunochemical evidence. To obtain structural information and to overcome limitations by the scarcity of endogenous mucin, we expressed a recombinant glycosylation probe corresponding to six MUC1 tandem repeats in four breast cancer cell lines. Comparative analyses of theO-glycan profiles were performed after hydrazinolysis and normal phase chromatography of 2-Aminobenzamide-labeled glycans. Except for a general reduction in the O-glycan chain lengths and a high density glycosylation, no common structural pattern was revealed. T47D fusion protein exhibits an almost complete shift from core 2 to core 1 expression with a preponderance of sialylated glycans. By contrast, MCF-7, MDA-MB231, and ZR75–1 cells glycosylate the MUC1 repeat peptide preferentially with core 2-based glycans terminating mostly with α3-linked sialic acid (MDA-MB231, ZR75–1) or α2/3-linked fucose (MCF-7). Endogenous MUC1 from T47D and MCF-7 cell supernatants revealed almost identicalO-glycosylation profiles compared with the respective recombinant probes, indicating that the fusion proteins reflected the authentic O-glycan profiles of the cells. The structural patterns in the majority of cells under study are in conflict with biosynthetic models of MUC1 O-glycosylation in breast cancer, which claim that the truncation of normal core 2-based polylactosamine structures to short sialylated core 1-based glycans is due to the reduced activity of core 2-forming β6-N-acetylglucosaminyltransferases and/or to overexpression of competitive α3- sialyltransferase.
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recombinant muc1 probe authentically reflects cell specific o glycosylation profiles of endogenous breast cancer mucin high density and prevalent core 2 based glycosylation
Journal of Biological Chemistry, 2002Co-Authors: Stefan Muller, Franzgeorg HanischAbstract:Knowledge about the O-linked glycan chains of tumor-associated MUC1 is primarily based on enzymatic and immunochemical evidence. To obtain structural information and to overcome limitations by the scarcity of endogenous mucin, we expressed a recombinant glycosylation probe corresponding to six MUC1 tandem repeats in four breast cancer cell lines. Comparative analyses of the O-glycan profiles were performed after hydrazinolysis and normal phase chromatography of 2-Aminobenzamide-labeled glycans. Except for a general reduction in the O-glycan chain lengths and a high density glycosylation, no common structural pattern was revealed. T47D fusion protein exhibits an almost complete shift from core 2 to core 1 expression with a preponderance of sialylated glycans. By contrast, MCF-7, MDA-MB231, and ZR75-1 cells glycosylate the MUC1 repeat peptide preferentially with core 2-based glycans terminating mostly with alpha 3-linked sialic acid (MDA-MB231, ZR75-1) or alpha 2/3-linked fucose (MCF-7). Endogenous MUC1 from T47D and MCF-7 cell supernatants revealed almost identical O-glycosylation profiles compared with the respective recombinant probes, indicating that the fusion proteins reflected the authentic O-glycan profiles of the cells. The structural patterns in the majority of cells under study are in conflict with biosynthetic models of MUC1 O-glycosylation in breast cancer, which claim that the truncation of normal core 2-based polylactosamine structures to short sialylated core 1-based glycans is due to the reduced activity of core 2-forming beta 6-N-acetylglucosaminyltransferases and/or to overexpression of competitive alpha 3- sialyltransferase.
Stefan Muller - One of the best experts on this subject based on the ideXlab platform.
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recombinant muc1 probe authentically reflects cell specific o glycosylation profiles of endogenous breast cancer mucin high density and prevalent core 2 based glycosylation
Journal of Biological Chemistry, 2002Co-Authors: Stefan Muller, Franzgeorg HanischAbstract:Abstract Knowledge about the O-linked glycan chains of tumor-associated MUC1 is primarily based on enzymatic and immunochemical evidence. To obtain structural information and to overcome limitations by the scarcity of endogenous mucin, we expressed a recombinant glycosylation probe corresponding to six MUC1 tandem repeats in four breast cancer cell lines. Comparative analyses of theO-glycan profiles were performed after hydrazinolysis and normal phase chromatography of 2-Aminobenzamide-labeled glycans. Except for a general reduction in the O-glycan chain lengths and a high density glycosylation, no common structural pattern was revealed. T47D fusion protein exhibits an almost complete shift from core 2 to core 1 expression with a preponderance of sialylated glycans. By contrast, MCF-7, MDA-MB231, and ZR75–1 cells glycosylate the MUC1 repeat peptide preferentially with core 2-based glycans terminating mostly with α3-linked sialic acid (MDA-MB231, ZR75–1) or α2/3-linked fucose (MCF-7). Endogenous MUC1 from T47D and MCF-7 cell supernatants revealed almost identicalO-glycosylation profiles compared with the respective recombinant probes, indicating that the fusion proteins reflected the authentic O-glycan profiles of the cells. The structural patterns in the majority of cells under study are in conflict with biosynthetic models of MUC1 O-glycosylation in breast cancer, which claim that the truncation of normal core 2-based polylactosamine structures to short sialylated core 1-based glycans is due to the reduced activity of core 2-forming β6-N-acetylglucosaminyltransferases and/or to overexpression of competitive α3- sialyltransferase.
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recombinant muc1 probe authentically reflects cell specific o glycosylation profiles of endogenous breast cancer mucin high density and prevalent core 2 based glycosylation
Journal of Biological Chemistry, 2002Co-Authors: Stefan Muller, Franzgeorg HanischAbstract:Knowledge about the O-linked glycan chains of tumor-associated MUC1 is primarily based on enzymatic and immunochemical evidence. To obtain structural information and to overcome limitations by the scarcity of endogenous mucin, we expressed a recombinant glycosylation probe corresponding to six MUC1 tandem repeats in four breast cancer cell lines. Comparative analyses of the O-glycan profiles were performed after hydrazinolysis and normal phase chromatography of 2-Aminobenzamide-labeled glycans. Except for a general reduction in the O-glycan chain lengths and a high density glycosylation, no common structural pattern was revealed. T47D fusion protein exhibits an almost complete shift from core 2 to core 1 expression with a preponderance of sialylated glycans. By contrast, MCF-7, MDA-MB231, and ZR75-1 cells glycosylate the MUC1 repeat peptide preferentially with core 2-based glycans terminating mostly with alpha 3-linked sialic acid (MDA-MB231, ZR75-1) or alpha 2/3-linked fucose (MCF-7). Endogenous MUC1 from T47D and MCF-7 cell supernatants revealed almost identical O-glycosylation profiles compared with the respective recombinant probes, indicating that the fusion proteins reflected the authentic O-glycan profiles of the cells. The structural patterns in the majority of cells under study are in conflict with biosynthetic models of MUC1 O-glycosylation in breast cancer, which claim that the truncation of normal core 2-based polylactosamine structures to short sialylated core 1-based glycans is due to the reduced activity of core 2-forming beta 6-N-acetylglucosaminyltransferases and/or to overexpression of competitive alpha 3- sialyltransferase.
Olga Gornik - One of the best experts on this subject based on the ideXlab platform.
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comparison of 2 Aminobenzamide procainamide and rapifluor ms as derivatizing agents for high throughput hilic uplc flr ms n glycan analysis
Frontiers in Chemistry, 2018Co-Authors: Toma Keser, Tamara Pavic, Gordan Lauc, Olga GornikAbstract:Rising awareness of the universal importance of protein N-glycosylation governs the development of further advances in N-glycan analysis. Nowadays it is well known that correct glycosylation is essential for proper protein function, which emanates from its important role in many physiological processes. Furthermore, glycosylation is involved in pathophysiology of multiple common complex diseases. In the vast majority of cases, N-glycosylation profiles are analyzed from enzymatically released glycans, which can be further derivatized in order to enhance the sensitivity of the analysis. Techniques wherein derivatized N-glycans are profiled using hydrophilic interaction chromatography (HILIC) with fluorescence (FLR) and mass spectrometry (MS) detection are now routinely performed in a high-throughput manner. Therefore, we aimed to examine the performance of frequently used labeling compounds -2-aminiobenzamide (2-AB) and procainamide (ProA), and the recently introduced RapiFluor-MS (RF-MS) fluorescent tag. In all experiments N-glycans were released by PNGase F, fluorescently derivatized, purified by HILIC solid phase extraction and profiled using HILIC-UPLC-FLR-MS. We assessed sensitivity, linear range, limit of quantification (LOQ), repeatability and labeling efficiency for all three labels. For this purpose, we employed in-house prepared IgG and a commercially available IgG as a model glycoprotein. All samples were analyzed in triplicates using different amounts of starting material. We also tested the performance of all three labels in a high-throughput setting on 68 different IgG samples, all in duplicates and 22 identical IgG standards. In general, ProA labeled glycans had the highest FLR sensitivity (15-fold and 4-fold higher signal intensities compared to 2-AB and RF-MS respectively) and RF-MS had the highest MS sensitivity (68-fold and 2-fold higher signal intensities compared to 2-AB and ProA, respectively). ProA and RF-MS showed comparable limits of quantification with both FLR and MS detection, whilst 2-AB exhibited the lowest sensitivity. All labeling procedures showed good and comparable repeatability. Furthermore, the results indicated that labeling efficiency was very similar for all three labels. In conclusion, all three labels are a good choice for N-glycan derivatization in high-throughput HILIC-UPLC-FLR-MS N-glycan analysis, although ProA and RF-MS are a better option when higher sensitivity is needed.
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Data_Sheet_1_Comparison of 2-Aminobenzamide, Procainamide and RapiFluor-MS as Derivatizing Agents for High-Throughput HILIC-UPLC-FLR-MS N-glycan Analysis.DOCX
2018Co-Authors: Toma Keser, Gordan Lauc, Tamara Pavić, Olga GornikAbstract:Rising awareness of the universal importance of protein N-glycosylation governs the development of further advances in N-glycan analysis. Nowadays it is well known that correct glycosylation is essential for proper protein function, which emanates from its important role in many physiological processes. Furthermore, glycosylation is involved in pathophysiology of multiple common complex diseases. In the vast majority of cases, N-glycosylation profiles are analyzed from enzymatically released glycans, which can be further derivatized in order to enhance the sensitivity of the analysis. Techniques wherein derivatized N-glycans are profiled using hydrophilic interaction chromatography (HILIC) with fluorescence (FLR) and mass spectrometry (MS) detection are now routinely performed in a high-throughput manner. Therefore, we aimed to examine the performance of frequently used labeling compounds −2-aminiobenzamide (2-AB) and procainamide (ProA), and the recently introduced RapiFluor-MS (RF-MS) fluorescent tag. In all experiments N-glycans were released by PNGase F, fluorescently derivatized, purified by HILIC solid phase extraction and profiled using HILIC-UPLC-FLR-MS. We assessed sensitivity, linear range, limit of quantification (LOQ), repeatability and labeling efficiency for all three labels. For this purpose, we employed in-house prepared IgG and a commercially available IgG as a model glycoprotein. All samples were analyzed in triplicates using different amounts of starting material. We also tested the performance of all three labels in a high-throughput setting on 68 different IgG samples, all in duplicates and 22 identical IgG standards. In general, ProA labeled glycans had the highest FLR sensitivity (15-fold and 4-fold higher signal intensities compared to 2-AB and RF-MS respectively) and RF-MS had the highest MS sensitivity (68-fold and 2-fold higher signal intensities compared to 2-AB and ProA, respectively). ProA and RF-MS showed comparable limits of quantification with both FLR and MS detection, whilst 2-AB exhibited the lowest sensitivity. All labeling procedures showed good and comparable repeatability. Furthermore, the results indicated that labeling efficiency was very similar for all three labels. In conclusion, all three labels are a good choice for N-glycan derivatization in high-throughput HILIC-UPLC-FLR-MS N-glycan analysis, although ProA and RF-MS are a better option when higher sensitivity is needed.
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high throughput isolation and glycosylation analysis of igg variability and heritability of the igg glycome in three isolated human populations
Molecular & Cellular Proteomics, 2011Co-Authors: Maja Pucic, Olga Gornik, Ana Knezevic, Jana Vidic, Barbara Adamczyk, Mislav Novokmet, Ozren Polasek, Sandra Suprahagoreta, Mark R Wormald, Irma RedzicAbstract:All immunoglobulin G molecules carry N-glycans, which modulate their biological activity. Changes in N-glycosylation of IgG associate with various diseases and affect the activity of therapeutic antibodies and intravenous immunoglobulins. We have developed a novel 96-well protein G monolithic plate and used it to rapidly isolate IgG from plasma of 2298 individuals from three isolated human populations. N-glycans were released by PNGase F, labeled with 2-Aminobenzamide and analyzed by hydrophilic interaction chromatography with fluorescence detection. The majority of the structural features of the IgG glycome were consistent with previous studies, but sialylation was somewhat higher than reported previously. Sialylation was particularly prominent in core fucosylated glycans containing two galactose residues and bisecting GlcNAc where median sialylation level was nearly 80%. Very high variability between individuals was observed, approximately three times higher than in the total plasma glycome. For example, neutral IgG glycans without core fucose varied between 1.3 and 19%, a difference that significantly affects the effector functions of natural antibodies, predisposing or protecting individuals from particular diseases. Heritability of IgG glycans was generally between 30 and 50%. The individual's age was associated with a significant decrease in galactose and increase of bisecting GlcNAc, whereas other functional elements of IgG glycosylation did not change much with age. Gender was not an important predictor for any IgG glycan. An important observation is that competition between glycosyltransferases, which occurs in vitro, did not appear to be relevant in vivo, indicating that the final glycan structures are not a simple result of competing enzymatic activities, but a carefully regulated outcome designed to meet the prevailing physiological needs.
Pauline M Rudd - One of the best experts on this subject based on the ideXlab platform.
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a comparative study of free oligosaccharides in the milk of domestic animals
British Journal of Nutrition, 2014Co-Authors: Simone Albrecht, Jonathan A Lane, Karina Marino, Khalid Ahmed Al Busadah, Stephen D Carrington, Rita M Hickey, Pauline M RuddAbstract:The present study was conducted to obtain a comprehensive overview of oligosaccharides present in the milk of a variety of important domestic animals including cows, goats, sheep, pigs, horses and dromedary camels. Using an analytical workflow that included ultra-performance liquid chromatography-hydrophilic interaction liquid chromatography with fluorescence detection coupled to quadrupole time-of-flight MS, detailed oligosaccharide libraries were established. The partial or full characterisation of the neutral/fucosylated, phosphorylated and sialylated structures was facilitated by sequencing with linkage- and sugar-specific exoglycosidases. Relative peak quantification of the 2-Aminobenzamide-labelled oligosaccharides provided additional information. Milk from domestic animals contained a much larger variety of complex oligosaccharides than was previously assumed, and thirteen of these structures have been identified previously in human milk. The direct comparison of the oligosaccharide mixtures reflects their role in the postnatal maturation of different types of gastrointestinal systems, which, in this way, are prepared for certain post-weaning diets. The potential value of animal milk for the commercial extraction of oligosaccharides to be used in human and animal health is highlighted.
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n and o glycosylation analysis of etanercept using liquid chromatography and quadrupole time of flight mass spectrometry equipped with electron transfer dissociation functionality
Analytical Chemistry, 2014Co-Authors: Stephane Houel, Pauline M Rudd, Mark Hilliard, Niaobh Mcloughlin, Jonathan P. Williams, Silvia Millan Martin, Weibin ChenAbstract:Etanercept is a highly glycosylated therapeutic Fc-fusion protein that contains multiple N- and O-glycosylation sites. An in-depth characterization of the glycosylation of etanercept was carried out using liquid chromatography/mass spectrometry (LC/MS) methods in a systematic approach in which we analyzed the N- and O-linked glycans and located the occupied O-glycosylation sites. Etanercept was first treated with peptide N-glycosidase F to release the N-glycans. The N-glycan pool was labeled with a 2-Aminobenzamide (2-AB) fluorescence tag and separated using ultraperformance liquid chromatography–hydrophilic interaction liquid chromatography (UPLC-HILIC). Preliminary structures were assigned using Glycobase. These assignments, which included monosaccharide sequence and linkage information, were confirmed by exoglycosidase array digestions of aliquots of the N-glycan pool. The removal of the N-glycans from etanercept facilitated the selective characterization of O-glycopeptides and enabled the O-glycans to...
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N- and O‑Glycosylation Analysis of Etanercept Using Liquid Chromatography and Quadrupole Time-of-Flight Mass Spectrometry Equipped with Electron-Transfer Dissociation Functionality
2014Co-Authors: Stephane Houel, Pauline M Rudd, Mark Hilliard, Niaobh Mcloughlin, Silvia Millan Martin, Jonathan P. Williams, Weibin ChenAbstract:Etanercept is a highly glycosylated therapeutic Fc-fusion protein that contains multiple N- and O-glycosylation sites. An in-depth characterization of the glycosylation of etanercept was carried out using liquid chromatography/mass spectrometry (LC/MS) methods in a systematic approach in which we analyzed the N- and O-linked glycans and located the occupied O-glycosylation sites. Etanercept was first treated with peptide N-glycosidase F to release the N-glycans. The N-glycan pool was labeled with a 2-Aminobenzamide (2-AB) fluorescence tag and separated using ultraperformance liquid chromatography–hydrophilic interaction liquid chromatography (UPLC-HILIC). Preliminary structures were assigned using Glycobase. These assignments, which included monosaccharide sequence and linkage information, were confirmed by exoglycosidase array digestions of aliquots of the N-glycan pool. The removal of the N-glycans from etanercept facilitated the selective characterization of O-glycopeptides and enabled the O-glycans to be identified. These were predominantly of the core 1 subtype (HexHexNAc O-structure) attached to Ser/Thr residues. α2→3,6,8,9 sialidase was used to remove the sialic acid residues on the O-glycans allowing the use of an automated LC/MSE protocol to identify the O-glycopeptides. Electron-transfer dissociation (ETD) was then used to pinpoint the 12 occupied O-glycosylation sites. The determination of N- and O-glycans and O-glycosylation sites in etanercept provides a basis for future studies addressing the biological importance of specific protein glycosylations in the production of safe and efficacious biotherapeutics
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the n glycosylation of classical swine fever virus e2 glycoprotein extracellular domain expressed in the milk of goat
Archives of Biochemistry and Biophysics, 2010Co-Authors: Raquel Montesino, Louise Royle, Raymond A Dwek, Pauline M Rudd, Jeovanis Gil, L Gonzalez, Yasser Zamora, D J Harvey, Jose A CremataAbstract:Classical swine fever virus (CSFV) outer surface E2 glycoprotein represents an important target to induce protective immunization during infection but the influence of N-glycosylation pattern in antigenicity is yet unclear. In the present work, the N-glycosylation of the E2-CSFV extracellular domain expressed in goat milk was determined. Enzymatic N-glycans releasing, 2-Aminobenzamide (2AB) labeling, weak anion-exchange and normal-phase HPLC combined with exoglycosidase digestions and mass spectrometry of 2AB-labeled and unlabeled N-glycans showed a heterogenic population of oligomannoside, hybrid and complex-type structures. The detection of two Man8GlcNAc2 isomers indicates an alternative active pathway in addition to the classical endoplasmic reticulum processing. N-acetyl or N-glycolyl monosialylated species predominate over neutral complex-type N-glycans. Asn207 site-specific micro-heterogeneity of the E2 most relevant antigenic and virulence site was determined by HPLC-mass spectrometry of glycopeptides. The differences in N-glycosylation with respect to the native E2 may not disturb the main antigenic domains when expressed in goat milk.
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separation of 2 Aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with 1 7 μm sorbent
Journal of Chromatography B, 2010Co-Authors: Joomi Ahn, Pauline M Rudd, Jonathan Bones, Martin GilarAbstract:Abstract Separation by hydrophilic interaction chromatography (HILIC) with fluorescence detection utilizing a sub-2 μm glycan column for the separation of 2-Aminobenzamide (2-AB) labeled N-linked glycans is described. The HILIC column packed with a 1.7 μm amide sorbent improves the peak capacity compared to a 3.0 μm HILIC column by a similar degree as observed in reversed-phase ultra-performance liquid chromatography (RP-UPLC). The results indicated that the optimal peak capacity was achieved at flow rate 0.2–0.5 mL/min. HILIC method transfer guidelines were shown to further enhance the resolution of glycans by changing initial gradient conditions, flow rate, column temperature, and different column lengths. Additionally, excellent resolution can be achieved in the separation of 2-AB labeled glycans released from fetuin, RNase B, and human IgG with a rapid analysis time.
Min Ji - One of the best experts on this subject based on the ideXlab platform.
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discovery bioactivity and docking simulation of vorinostat analogues containing 1 2 4 oxadiazole moiety as potent histone deacetylase inhibitors and antitumor agents
Bioorganic & Medicinal Chemistry, 2015Co-Authors: Kwon Ho Hong, Xiaoqing Wu, Xi Zong, Lushen Li, Junqing Chen, Peng Wang, Bo Chen, Gaoxing Zhou, Min JiAbstract:In our study, three series of hydroxamate, 2-Aminobenzamide, and trifluoromethyl ketone analogues have been designed and synthesized. The synthesized compounds were investigated for their in vitro antiproliferative activities using the MTT-based assay against three human cancer cell lines including A549, NCI-H661, and U937. Most analogues exhibited higher antiproliferative activities against human acute myeloid leukemia cell U937 than the other two human lung cancer cell lines. Furthermore, the compounds were examined against HDAC1, 2, and 8 isoforms. Docking study of compounds 6h, 9b, and 10a suggested that they might bind tightly to the binding pocket of HDAC2 and/or HDAC8. The results suggest that these compounds might have potential as lead compounds for the development of anti-tumor drugs with HDACs inhibitory activities.
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discovery and preliminary evaluation of 2 Aminobenzamide and hydroxamate derivatives containing 1 2 4 oxadiazole moiety as potent histone deacetylase inhibitors
European Journal of Medicinal Chemistry, 2015Co-Authors: Kwon Ho Hong, Xiaoqing Wu, Xi Zong, Lushen Li, Junqing Chen, Min JiAbstract:Abstract Using Entinostat as a lead compound, 2-Aminobenzamide and hydroxamate derivatives have been designed and synthesized. The entire target compounds were investigated for their in vitro antiproliferative activities using the MTT-based assay against five human cancer cell lines including U937, A549, NCI-H661, MDA-MB-231 and HCT116. 2-Aminobenzamide series of compounds (10a–10j) demonstrated the most significant inhibition against human acute monocytic myeloid leukemia cell line U937, but no or poor activities against two human lung cancer cell lines. Furthermore, the target compounds were screened for their inhibitory activities against HDAC 1, 2, and 8. 2-Aminobenzamide derivatives (10) manifested a higher selectivity for HDAC 1 over HDAC 2, but were not active against HDAC 8. In contrast, most hydroxamate derivatives (11) inhibit HDAC 8 with lower IC50 values than SAHA and Entinostat. Docking study with selected compounds 10f and 11a revealed that the compounds might bind tightly to the binding pockets in HDAC 2 and HDAC 8, respectively. The results suggest that they may be promising lead compounds for the development of novel anti-tumor drug potentially via inhibiting HDACs.
