Enhancer of zeste homolog 2 (EZH2) tri-methylates histone 3 at position lysine 27 (H3K27me3). Overexpression and gain-of-function mutations in EZH2 are regarded as oncogenic drivers in lymphoma and other malignancies due to the silencing of tumor suppressors and differentiation genes. EZH2 inhibition is sought to represent a good strategy for tumor therapy. In this study, we treated Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) cell lines with 3-deazaneplanocin—A (DZNep), an indirect EZH2 inhibitor which possesses anticancer properties both in-vitro and in-vivo. We aimed to address the impact of the lymphoma type, EZH2 mutation status, as well as MYC, BCL2 and BCL6 translocations on the sensitivity of the lymphoma cell lines to DZNep-mediated apoptosis. We show that DZNep inhibits proliferation and induces apoptosis of these cell lines independent of the type of lymphoma, the EZH2 mutation status and the MYC, BCL2 and BCL6 rearrangement status. Furthermore, DZNep induced a much stronger apoptosis in majority of these cell lines at a lower concentration, and within a shorter period when compared with EPZ-6438, a direct EZH2 inhibitor currently in phase II clinical trials. Apoptosis induction by DZNep was both concentration-dependent and time-dependent, and was associated with the inhibition of EZH2 and subsequent downregulation of H3K27me3 in DZNep-sensitive cell lines. Although EZH2, MYC, BCL2 and BCL6 are important prognostic biomarkers for lymphomas, our study shows that they poorly influence the sensitivity of lymphoma cell lines to DZNep-mediated apoptosis. EZH2 is) levels . This in) levels –28—and t2.Seven diffuse large B-cell lymphoma (DLBCL) cell lines HT (ACC-567), WSU-DLCL-2 (ACC-575), SU-DHL-10 (ACC-576), CARNAVAL (ACC-724), U-2932 R1, U-2932 R2, KARPAS-422 (ACC-32), and five Burkitt lymphoma (BL) cell lines BLUE-1 (ACC-594), CA-46 (ACC-73), DG-75 (ACC-83), BL-2 (ACC-625), BL-41 (ACC-160) were used. All cell lines were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Braunschweig, Germany. U-2932 R1 and U-2932 R2 sub clones were donated by Dr. Hilmar Quentmeier [DZNep  was dissolved in sterile water, and EPZ-6438  was dissolved in dimethyl sulfoxide (DMSO)  as recommended by the manufacturer. Both drugs were diluted with the respective solvents and stored at -20°C until used for the treatment of cell lines.5 cells from the well-mixed culture were washed twice with cell staining buffer . The supernatant was discarded and the cells stained with a mixture of annexin V (Ann V) binding buffer , propidium iodide (PI)  and allophycocyanin (APC) annexin V  at a ratio of 20:2:1 respectively. After a 10-minute incubation step at room temperature, the number of apoptotic cells was calculated using the BD Accuri C6 software . The fraction of cells gated to indicate apoptosis include the Ann V/PI-positive cells.For measurement of apoptotic cells, flow cytometry using the BD Accuri C6 flow cytometer  was performed. Briefly, 3 x 105 cells / ml in cell culture flasks and treated with 5 μM DZNep, or without DZNep. The number of vital cells were counted every 24 hours, 48 hours and 72 hours using flow cytometry with appropriate gating to exclude apoptotic and dead cells.The respective cell lines were seeded at an initial cell density of 2 x 1050 was defined as the concentration of DZNep required to decrease cell viability by 50%. Cell lines were treated in 6-well cell culture plates with increasing concentrations of DZNep ranging from 0.5 μM until 10 μM. The percentage of annexin V/PI positive (non-viable) cells was measured afterwards. A plot of the percentage of non-viable cells versus concentration of DZNep was made and the IC50 was calculated for each cell line using linear regression.The IC6 cells were harvested and washed with 1x Gibco Dulbecco’s phosphate buffered saline (DPBS) . Whole cell lysis was done with 200 μl RIPA buffer  and 1x Complete protease inhibitor cocktail . These were added to the cells and the mixture incubated for 30 minutes on ice. Afterwards, 2 cycles of high power sonication with Bioruptor Sonicator  was applied onto the cells. A centrifugation step for 15 minutes at 13,000 revolutions per minute was performed and the supernatant was collected and used for protein concentration determination. The rest of the lysate was stored at -80°C prior to use. The protein concentration was measured using the Pierce Bicinchoninic acid (BCA) protein assay Kit , following the manufacturers protocol. 10–15 μg of the protein lysate was separated on a 12% Expedeon RunBlue SDS protein gel . The protein was transferred onto a 0.2 μM Immobilon-P-membrane  by electroblotting. After transfer, the membrane was blocked in 5% milk solution (dissolved in PBST) for 1 hour. The membranes were incubated with the primary antibodies overnight at 4°C. After washing the membrane 3 times for 5 minutes in PBST, the secondary antibody was applied. Incubation of the membrane with the secondary antibody was done at room temperature for 1 hour. Afterwards, the membranes were once again washed in PBST three times for 5 minutes. Immobilion Forte Western HRP substrate  was added onto the blot to aid chemiluminiscence. Signals were detected using the FUSION-SL-3500 system . Image analysis and signal quantification was done using the FUSION-CAP Software . Primary antibodies used for Western blot include anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody , anti-EZH2 antibody , anti- H3K27me3 antibody , and anti-cleaved PARP antibody  . The secondary antibody used was anti-rabbit HRP-conjugated  .3 x 10Genomic DNA was extracted from cells using QIAamp DNA Mini Kit, and RNA was isolated from cells using the RNeasy Midi Kit  following the recommendations of the manufacturer. For cDNA synthesis, total RNA was treated with DNase I  for 30 minutes at 37°C. To the DNA-digested product, 50 mM EDTA was added and incubated for 10 minutes at 65°C. Total RNA was reverse transcribed with a T3 thermocycler  to cDNA using TaqMan Reverse Transcription reagents and random hexamer primers . Cycling conditions used include primer annealing at 25°C for 10 minutes, DNA polymerization at 37°C for 30 minutes and finally reverse transcriptase deactivation at 95°C for 5 minutes. The synthesized cDNA was stored at -20°C pending further use.5’-TCCCCAGTCCATTTTCACC-3’ and 5’-TCATTTCCAATCAAACCCACAG-3’ respectively (product length = 256 base pairs). For detection of EZH2 point mutations at the RNA (cDNA) level, the forward and reverse primer sequence utilized for Sanger sequencing include TGACCTCTGTCTTACTTGTGGAG-3’5’- and GCAGTTTGGATTTACCGAATG-3’5’- respectively. This primer sequence covers the EZH2 mutation hotspots on exon 16 and 18, with a product length of 340 base pairs. PCR was performed on the ProFlex PCR system Thermocycler . 10 μl of the respective PCR products was mixed with 2 μl 6x Gel loading dye  and loaded onto 7% polyacrylamide gels. 6 μl Gene Ruler low range DNA ladder  was also loaded onto the gel. For the run, 1x Tris-borate-EDTA (TBE) buffer was used, and gels were set to run for 30 minutes at 150 Volts. The gel image was developed upon staining with ethidium bromide , and the image was captured using the Gel Doc 2000 . The PCR products were cleaned using the Wizard SV Gel and PCR Clean-Up System  following the manufacturers instruction. The concentration of the cleaned-up PCR product was measured using the NanoDrop ND-1000 . Sanger sequencing was done by LGC Genomics GmbH, Berlin, Germany.EZH2 primers were designed using the NCBI Primer BLAST software . The forward and reverse primer sequence for the EZH2 genomic region of exon 16 include FISH was performed on formalin-fixed paraffin-embedded (FFPE) tissue sections and cytospins of cell lines as already described , 31. ProStatistical analysis was done using the GraphPad Prism 5 software . Statistical significance between groups was determined using the Mann-Whitney U test (two-tailed) and p values < 0.05 were considered significant.The functional significance of EZH2 overexpression and gain-of-function mutations in cancer has been extensively studied –40. Its This heterozygous Tyr646 point mutation has also been demonstrated by other reports investigating the role of these gain-of-function mutations in primary tumor patient samples , 42–45. To establish optimal conditions for growth of the cell lines with and without DZNep pressure, and to show when the cell lines begin to exhibit an apoptotic phenotype, we made concentration- and time-course experiments. We sought a situation where one could optimally measure apoptosis upon DZNep treatment, while excluding external apoptosis-inducing factors such as overcrowding of the cells in the culture flask.We reveal that cell death caused by DZNep is concentration-dependent , increasProlonged treatment of cell lines past 72 hours was not recommended because of overgrowth of the controls past the recommended maximum cell density provided by the DSMZ. Our results corroborate similar concentration and time-dependence of apoptosis mediated by DZNep in colon cancer cell lines  and non-50 experiment. The IC50 is the concentration of DZNep that decreases the percentage of viable cells of a given cell line by 50%. From our data , BCL2 (18q21) and BCL6 (3q27) translocations. For the latter purpose, we performed FISH analysis with the respective fluorescence-labeled probes on the cell lines. A summary of our findings is shown in Our results reveal no particular trend associating the DZNep sensitivity or resistance of the cell lines with the type of lymphoma, or translocations involving the genes for MYC, BLC2 or BCL6. This shows that the lymphoma type and these biomarkers are poor indicators of the effectiveness of DZNep in inducing apoptosis in B-cell lymphomas.: NCT02601950). First, we treated 2 EZH2- mutated  and 2 EZH2 wild-type (HT and BL-41) cell lines with 5 μM DZNep and 5 μM EPZ-6438 for 72 hours, and determined the percentage of apoptotic cells. We showed that DZNep causes apoptosis in both wild-type and EZH2-mutated cell lines, showing no preference for each case. In addition, DZNep caused obvious apoptosis (p = 0.1) of DZNep-sensitive cell lines under similar conditions in comparison with EPZ-6438  which is currently in phase II clinical trials for patients with solid tumors EPZ-6438 . We alsoEPZ-6438 . For theAs with cell lines of other cancer entities such as chondrosarcoma , breast Since some cell lines remained resistant to DZNep despite its strong apoptotic capacity, further investigation into the molecular mechanism of resistance to DZNep is recommended, as this would enable the identification of biomarkers that could be of therapeutic relevance for EZH2 inhibition by DZNep.Our work reveals the anti-tumor properties of DZNep and shows its strong apoptotic effects in aggressive B-cell lymphoma cell lines. We furthermore provide evidence that the increased EZH2 function mediated by mutated EZH2 does not decrease the ability of DZNep to inhibit H3K27me3 and induce apoptosis in the cell lines. In addition, although MYC, BCL2 and BCL6 rearrangements are important prognostic markers for patients with DLBCL, the sensitivity of lymphoma cell lines to DZNep-mediated apoptosis is independent of these rearrangements. Finally, the type of lymphoma, BL or DLBCL is not predictive for the success of DZNep treatment. We are currently working on the identification of alternative biomarkers.S1 Fig(PDF)Click here for additional data file.S2 FigFISH data from lymphoma cell lines showing the presence (+) or absence (-) of (A) MYC break, (B) BCL2 rearrangement and (C) BCL6 translocations. Presence of MYC translocation in SU-DHL-10, DG-75 and CA-46 is documented in the DSMZ data sheet for the cell lines .(PDF)Click here for additional data file.


Lutzomyia longipalpis and the biting midge Culicoides sonorensis, and are actively engaging with collaborators to obtain starting material for primary cell cultures from other midge species, mites, tsetse flies and bees. Outposts of the Tick Cell Biobank will be set up in Malaysia, Kenya and Brazil to facilitate uptake and exploitation of cell lines and associated training by scientists in these and neighbouring countries. Thus the Tick Cell Biobank will continue to underpin many areas of global research into biology and control of ticks, other arthropods and vector-borne viral, bacterial and protozoan pathogens.Tick cell lines are increasingly used in many fields of tick and tick-borne disease research. The Tick Cell Biobank was established in 2009 to facilitate the development and uptake of these unique and valuable resources. As well as serving as a repository for existing and new ixodid and argasid tick cell lines, the Tick Cell Biobank supplies cell lines and training in their maintenance to scientists worldwide and generates novel cultures from tick species not already represented in the collection. Now part of the Institute of Infection and Global Health at the University of Liverpool, the Tick Cell Biobank has embarked on a new phase of activity particularly targeted at research on problems caused by ticks, other arthropods and the diseases they transmit in less-developed, lower- and middle-income countries. We are carrying out genotypic and phenotypic characterisation of selected cell lines derived from tropical tick species. We continue to expand the culture collection, currently comprising 63 cell lines derived from 18 ixodid and argasid tick species and one each from the sand fly  In recein vitro. Cell lines have also been established from moulting larval (developing nymph) Amblyomma variegatum and moulting nymphal (developing adult) Rhipicephalus appendiculatus and Rhipicephalus evertsi. Specific tick organs such as midgut, salivary glands, haemocytes or ovaries have not, as yet, yielded cell lines despite numerous attempts to maintain and propagate them in vitro  at https://www.ncbi.nlm.nih.gov/bioproject/238805 and transcriptomic and proteomic datasets generated in tick-pathogen interaction studies , Minnesota and Texas A&M, with 3 years’ funding from the Wellcome Trust, to create a repository for existing tick cell lines, a resource to support their successful dissemination worldwide and a facility for establishment of new tick cell lines. At that time we housed 47 continuous tick cell lines originally established in five laboratories  of which 36 were sufficiently robust to be distributed. From the start, an important part of our remit was to provide training in tick cell line maintenance in order to facilitate successful transfer to, and application in, recipient laboratories; latterly this was extended to include training in primary tick cell and organ culture and cell line establishment.Hyalomma dromedarii, R. evertsi, R. appendiculatus and R. microplus and the argasid species Argas reflexus . Following published protocols -9-methylgermacrene-B , tsetse flies (Glossina spp.), UK mosquitoes (Culex and Aedes spp.), bumble bees (Bombus spp.) and honey bees (Apis mellifera).Another important aspect of the Tick Cell Biobank’s current remit is to establish, house and distribute cell lines derived from “neglected” arthropod species of importance as pathogen vectors, agricultural pests or pollinators. The Biobank already houses the only widely-used biting midge cell line KC, derived from norensis , and is rotocols , we havegipalpis ; the paracrene-B . As and 6R. microplus  in the tropics and sub-tropics. Direct and indirect losses are incurred through costs of tick and tick-borne disease control , reduced productivity, mortality and time expended on control . Acariciicroplus . Major tin vivo studies to make a significant contribution to local and global tick-related problems. Currently it is difficult and expensive for LMIC researchers to obtain and maintain existing TCL to support their research, as the cells must be obtained from UK or USA and local training is unavailable. Resources for research on pathogens transmitted by other arthropods such as sand flies, midges, mites and fleas are even more difficult to obtain or non-existent.Tick cell lines are increasingly applied in many aspects of research into control of ticks and tick-borne diseases of livestock and humans, particularly those of importance in LMIC. However, most of this research is carried out in high-income countries (HIC) in Europe, North America and the Asia-Pacific region . This conundrum is illustrated in To address this deficit, we are establishing outposts of the Tick Cell Biobank in Malaysia, Kenya and Brazil, with the aim of facilitating access to tick cell lines for LMIC scientists, and increasing uptake and application of this technology in their research. The outposts will serve not only their host institute and country, but will disseminate materials and expertise in the surrounding regions of, respectively, South-East Asia, Africa and South America. Each outpost will house a panel of the most popular cell lines, plus some with specific regional relevance, and will distribute cell lines and locally-tailored training in their maintenance. Where possible, they will also attempt to establish novel cell lines from local ticks and other arthropods, building on preliminary studies in Malaysia  and Braz7Tick cell and tissue culture has progressed a long way since the first short-term cultures carried out in the 1950s and 1960s (reviewed by


Self-assembly into oligomers and fibrils during the process of aggregation by hIAPP can lead to failure and depletion of β-cells. Studies have shown that some critical regions of hIAPP might contribute to the aggregation. Thus, many studies focused on finding the effective molecules, especially the short-peptide inhibitors, that bind to these regions and disrupt the aggregation of hIAPP. In the present study, a novel pentapeptide inhibitor Phe-Leu-Pro-Asn-Phe (FLPNF) was designed and its effectiveness on the inhibition of the formation of amyloid deposits was examined.  The presence of amyloid deposits of human islet amyloid polypeptide (hIAPP) in islet  The binding mode between FLPNF and hIAPP was performed using molecular docking. The effectiveness of FLPNF on inhibiting hIAPP amyloid aggregation was tested by Thioflavin T (ThT) staining. Furthermore, negative stain electron microscopy was used to observe hIAPP fibrils. A biolayer interferometry analysis was used to identify the interaction between FLPNF and hIAPP. In addition, the cytotoxicity toward INS-1 cells was tested by a cell proliferation assay. in vitro. Coincubation of FLPNF with hIAPP decreased the amount of hIAPP fibrils. Furthermore, a direct interaction between FLPNF and hIAPP was confirmed. FLPNF could also decrease the cytotoxic effect of hIAPP.  FLPNF was predicted to have a compact conformation to bind at the site of hIAPP. FLPNF strongly inhibited the amyloid aggregation of hIAPP at a 10 : 1 molar ratio  The novel pentapeptide inhibitor FLPNF was constructed and inhibited the aggregation through direct binding to hIAPP. It is considered a suitable inhibitor for hIAPP amyloid deposit formation. It igranules . In physgranules  and alsogranules . Monomer[β-cells –12. Lackgregates , 14. On gregates  and islegregates , 16.β-sheet structure [β-sheet aggregations. Studies have shown that sequence 8-20 or 30-37 of hIAPP is capable of forming fibrils itself [π-π stacking effect of aromatic residues plays a key role in causing aggregation of hIAPP [β) in Alzheimer's disease. These could inhibit the β-sheet aggregations of Aβ and hence were named as beta-sheet breaker peptides [Since hIAPP self-assembles to form amyloid deposits containing parallel tructure , it has s itself , and so s itself , 20. Stuof hIAPP , 22. In peptides , 24. Thiin vitro [in vivo [β-strand to prevent self-assembly [β-sheet protein deposits and changes the optical properties [So far, only few studies have examined short-peptide inhibitors for the inhibition of hIAPP. Peptides such as SNNFGA, GAILSS, NYGAILSS, and NFGAILPP can inhibit hIAPP aggregation in vitro , 26, whi[in vivo . In the assembly , 29. Meaassembly  and was operties , which tPeptides hIAPP, FLPNF, and NFGAIL were synthesized by ChinaPeptide Co. Ltd. . The synthetic peptides were solubilized in DMSO to make stock solutions with a final concentration of 10 mM hIAPP, 100 mM FLPNF, and 100 mM NFGAIL. The stock solutions were aliquoted and stored at -80°C according to the instructions.http://www.rcsb.org/). The 3D structure of FLPNF was built by the PyMOL 1.7.6 package (http://www.pymol.org/). The AutoDockTools 1.5.6 package [A molecular docking study was performed to investigate the binding mode between FLPNF and hIAPP using Autodock Vina 1.1.2 . The thr package , 33 was μL of each stock solution of hIAPP and FLPNF was added to 100 μL of PBS , and this group was set as “hIAPP+FLPNF.” Similarly, 0.1 μL stock solution of hIAPP and NFGAIL added to 100 μL of PBS (containing 20 μM ThT) was set as “hIAPP+NFGAIL.” Meanwhile, 0.1 μL stock solution of hIAPP with additional 0.1 μL DMSO was added to 100 μL PBS (containing 20 μM ThT), and this was set as “hIAPP.” The PBS solution with 20 μM ThT and 0.2 μL DMSO was set as the control. The plates were incubated at 37°C for 0, 12, 24, and 48 hours. Finally, the fluorescence image was detected by an XDY-100 fluorescence microscope . Subsequently, the fluorescence intensity values of each group were measured by a Fluoroskan Ascent FL luminescence meter  using an excitation λ of 435 nm and an emission λ of 485 nm. At each time course, the fluorescence intensity values of the control group were set as 1, while the values of test groups relative to the control group were set as fluorescence intensity (A.U.) and used for statistical analysis. Each experiment was repeated thrice.ThT  was used to stain the amyloid deposits of hIAPP in 96-well plates. A volume of 0.1 μM) of FLPNF or NFGAIL were added into PBS with 10 μM hIAPP at the same conditions mentioned above to verify their inhibitory effects. The fluorescence images and the fluorescence intensity values of each group were collected after incubating for 24 hours. Each experiment was repeated thrice.Different concentrations , in combination with 100 μM FLPNF (hIAPP+FLPNF), or in combination with 100 μM NFGAIL (hIAPP+NFGAIL) was incubated for 24 hours at 37°C. A volume of 10 μL of each sample was placed on mesh copper grids  and was covered by a carbon-stabilized formvar film. After 3 min, excess fluid was removed, and the grids were stained with 2% phosphotungstic acid for 3 min. Samples were analyzed using a HT7700 transmission electron microscope .TEM was used to directly observe the relative density and morphological changes of hIAPP amyloid fibrils. PBS solution containing 10 μM) was performed for 300 sec. This was followed by a 600 sec dissociation phase in dissociation buffer (50% DMSO+0.05% PBST) in PBS. A response unit represented the bonding height on the biosensor. The association and dissociation curves were analyzed and constructed by Octet K2 biolayer interferometry.Interaction between FLPNF and hIAPP was studied using Octet K2 biolayer interferometry . Experiments were performed by Shuangtian Biotech Co. Ltd. FLPNF were reconstituted in PBS (pH = 7.4) and immobilized on aminopropylsilane (APS) biosensors. Following 30 sec equilibration of immobilized FLPNF in PBS, the interaction with the target protein hIAPP . The cells were incubated with fresh medium containing 10 μM hIAPP alone, 10 μM hIAPP with 100 μM FLPNF, or 10 μM hIAPP with 100 μM NFGAIL for additional 24 hours. The untreated control cells received equal fresh medium. The medium of each group contained 0.2 μL DMSO, equal to 0.2% DMSO. Consequently, 10 μL MTS solution was added to each well and incubated for additional 2 hours. The optical density (OD) values were measured at 490 nm using a PowerWave XS2 microplate spectrophotometer . Each experiment was repeated thrice.INS-1 cells were purchased from BioLeaf Biotech Co. Ltd. . INS-1 cells were maintained as described previously . The INSt-test was performed for statistical analysis. P value < 0.05 was considered to be statistically significant.GraphPad Prism 7.0 software  was used for statistical analysis. Data are presented as means ± standard deviations (SD). Student's π interactions with the residues Lys-1 and Arg-11 of hIAPP, while the side chain of the residue Phe-5 of FLPNF formed a π-π stacking interaction with the residue Phe-15 of hIAPP. Importantly, two hydrogen bond interactions were observed between the residues Asn-4 and Phe-5 of FLPNF and the residues Asn-31 (bond length: 2.3 Å) and Arg-11 (bond length: 2.6 Å) of hIAPP, respectively, which were the main interaction between them , a gradual increase in flocculence and strip-like green fluorescence was observed. Meanwhile, the fluorescence signal was similar to that of the hIAPP group after adding NFGAIL (100 μM) at each time point. In addition, a strong reduction of the fluorescence signal was observed at 24 and 48 hours after the addition of FLPNF (100 μM). There was no green fluorescence observed in the control group at any time (The ability of the peptides FLPNF and NFGAIL to inhibit hIAPP aggregation in PBS was examined by the ThT fluorescence assay. After 12-hour incubation with hIAPP (10 any time .μM), followed by incubation with hIAPP, decreased the fluorescence intensity compared to hIAPP alone at 12, 24, and 48 hours. The fluorescence signal was significantly lowered in the hIAPP+FLPNF group compared to the hIAPP group at 24 h (P < 0.05) and 48 h (P < 0.01). The fluorescence intensity did not decrease in the hIAPP+NFGAIL group compared with the hIAPP group  of FLPNF, followed by incubation with 10 μM hIAPP for 24 hours at 37°C, the green fluorescence signal as observed by a fluorescence microscope was gradually decreased (μM groups were significantly lowered compared to that in the 0 μM group (P < 0.05)  . On the  Figures .μM) alone in PBS demonstrated typical dense fibrils by TEM after 24 hours, while the addition of FLPNF (100 μM) reduced the amounts of fibril formation. On the contrary, the addition of NFGAIL (100 μM) did not reduce or even increase the amount of the fibril formation observed by naked eyes  to INS-1 cells significantly decreased the cell viability compared to the control group (P < 0.001). The presence of FLPNF (100 μM) with hIAPP demonstrated a significant improvement in cell viability compared to the hIAPP group (P < 0.05). Moreover, FLPNF alone showed no direct effect on the viability of INS-1 cells. Adding NFGAIL (100 μM) showed no effect of reducing the cytotoxic effects of hIAPP , and the molecular weights of NYGAILSS and NFGAILPP were large . D-ANFLVH could reduce the islet amyloid accumulation in vivo with good characteristics [There were only few studies that investigated the effective peptide inhibitors for the treatment of hIAPP aggregation NFGAILPP , 26. ButFLPNF showed an inhibitory effect on hIAPP amyloid deposit formation by ThT staining. Also, TEM was used to observe the hIAPP fibrils to confirm the effect of FLPNF, where the fibrils were less numerous but appeared to be more thickened when FLPNF was added. This suggested that the interaction with hIAPP altered normal fibril assembly and warranted further investigation. The obtained results showed that FLPNF could reduce hIAPP aggregation but still could not completely prevent the formation of fibrils. In the early research, SNNFGA and GAILSS could not completely prevent the formation of fibrils of hIAPP on TEM images . So, it π-π stacking effect of aromatic residues plays a key role in aggregation of hIAPP [π interactions and the π-π stacking interaction between FLPNF and hIAPP might disrupt the self-assembly of hIAPP and were consistent with our original principles of designing a short-peptide inhibitor. Subsequently, biolayer interferometry was used to confirm that there was a direct interaction between them. Previous studies have not performed experiments to verify the mechanism of short-peptide inhibitors, but we provided experimental evidence and produced an ideal treatment strategy for hIAPP aggregation. Further research is still needed to confirm the specific interaction mode between FLPNF and hIAPP. Moreover, the results also showed that the residues 11-15 of hIAPP had a remarkable effect on fibrillogenesis.Since there was no other factor involved in the PBS environment, we speculated that the mechanism of FLPNF inhibiting hIAPP aggregation occurred through binding to key regions of hIAPP. The molecular docking simulations gave us rational explanation of the interactions between FLPNF and hIAPP, which provided valuable information for the study of the mechanism of action between them at the molecular level. Since the of hIAPP , 22, theμM hIAPP and were almost completely dead after 48 hours. In future, we plan to construct the INS-1 cell line expressing hIAPP to further study the function of peptide inhibitors. Further investigation regarding the ways of FLPNF inhibition on hIAPP aggregation in cell lines is warranted. Peptides may be used in vivo by intraperitoneal [in vivo in the future.FLPNF decreased the cytotoxic effect of hIAPP and then improved the cell viability of rat insulinoma cells. Meanwhile, FLPNF alone had no direct effect on cell viability toward INS-1 cells. This result remained interesting as FLPNF is a small and nontoxic compound and can be used for the inhibition of hIAPP aggregation. Nonetheless, the cell viability was still lower when hIAPP was incubated with FLPNF at a 1 : 10 ratio compared to the control group, possibly suggesting that the small cytotoxic aggregates could still be formed in the presence of FLPNF. These findings can be important for the refinement of future inhibitory molecules. In addition, we only measured the viability of cells within 24 hours because INS-1 cells were rapidly dying after 24 hours of incubation with 10 ritoneal  or intraritoneal  injectioin vitro is predicted as 1.1 hours by the software . However, our results showed that FLPNF still had an inhibitory effect at 48 hours in vitro. In the future, the half-life and the inhibitory effect of FLPNF in vivo need to be verified. Moreover, we also consider modifying this peptide to improve its stability, such as modification by PEGylation [in vivo investigations are needed.There are still some limitations about the pentapeptide; the half-life Gylation , 42. Thiin vitro and partially protecting INS-1 cells from the cytotoxic effect of hIAPP. Our research laid a foundation for future experiments in cell lines and in rat models. Nonetheless, the inhibitory effect needs to be further refined. The present study provided new insights into potential therapeutic compounds for the treatment of hIAPP aggregation.Taken together, our results have demonstrated that the optimal designed pentapeptide FLPNF is capable of inhibiting hIAPP fibril formation


Data from the Repository for Molecular Brain Neoplasia Data (REMBRANDT) dataset also showed the co-expression of choline transporters, choline acetyltransferase, and vesicular acetylcholine transporters, suggesting that GBMs express all the proteins required for ACh synthesis and release. These findings identify ACh as a modulator of GBM behavior and posit that GBMs may utilize ACh as an autocrine signaling molecule.Grade IV astrocytomas, or glioblastomas (GBMs), are the most common malignant primary brain tumor in adults. The median GBM patient survival of 12–15 months has remained stagnant, in spite of treatment strategies, making GBMs a tremendous challenge clinically. This is at least in part due to the complex interaction of GBM cells with the brain microenvironment and their tendency to aggressively infiltrate normal brain tissue. GBMs frequently invade supratentorial brain regions that are richly innervated by neurotransmitter projections, most notably acetylcholine (ACh). Here, we asked whether ACh signaling influences the biology of GBMs. We examined the expression and function of known ACh receptors (AChRs) in large GBM datasets, as well as, human GBM cell lines and patient-derived xenograft lines. Using RNA-Seq data from the “The Cancer Genome Atlas” (TCGA), we confirmed the expression of AChRs and demonstrated the functionality of these receptors in GBM cells with time-lapse calcium imaging. AChR activation did not alter cell proliferation or migration, however, it significantly increased cell invasion through complex extracellular matrices. This was due to the enhanced activity of matrix metalloproteinase-9 (MMP-9) from GBM cells, which we found to be dependent on an intracellular calcium-dependent mechanism. Consistent with these findings, AChRs were significantly upregulated in regions of GBM infiltration in situ (Ivy Glioblastoma Atlas Project) and elevated expression of muscarinic AChR M Glioblastomas (GBMs) are the most common primary malignant brain tumors in humans, occurring in approximately 13,000 people annually in the United States . This poThe cellular origin of GBMs has long been a point of contention, with debate centering on whether these tumors are the result of the dedifferentiation of mature cells or the accumulation of oncologic mutations in neural stem cells (NSCs) . Severalc− cystine–glutamate transporter to elevate peritumoral glutamate levels to excitotoxic levels for surrounding neurons i) as a readout of AChR activity. Increases in [Ca2+]i occur upon activation of both the mAChRs and nAChRs via different signaling cascades i lead to elevated release and subsequent MMP activity. First, we used the Repository of Molecular Brain Neoplasia Data (REMBRANDT) dataset to compare mRNA expression of MMP-2 and MMP-9 in non-tumor control and GBM samples  activity occurred with AChR stimulation d,e. Howe2+ increases during AChR stimulation played a role in MMP-9 activity, we used the Ca2+ chelator BAPTA-AM to prevent increases in [Ca2+]i. Following a 30 min BAPTA-AM incubation, the U251 and PDX14 lines were stimulated with ACh and the conditioned-cell medium were collected after 6 h and MMP activity was quantified with gelatin zymography. With [Ca2+]i chelated, mature MMP-9 activity was significantly reduced and pro- MMP-9 was not present in the U251 and PDX14 cells  to confirm that MMP-9 was only responsible for the increased invasion observed with AChR stimulation. In the U251 and PDX14 lines, we found that MMP-9 inhibition led to a significant decrease in invasion without the addition of AChR agonists, but with the addition of AChR agonists, no significant difference was observed in comparison to the control a,b. ThesCHRNA7, CHRNB2, CHRM1, and CHRM3 in these areas  database , we compse areas . These fOur findings suggest that ACh is an important signaling molecule in GBM biology. Many cells besides cholinergic neurons are capable of synthesizing ACh, including epithelial cells in the lungs, endothelial cells, and various immune cells ,44,45,46https://gdoc.georgetown.edu/gdoc/) i upon activation i. For this reason, we cannot conclude definitively that these subtypes of mAChRs are functional via this method. Furthermore, we are limited to observing a collective response of the muscarinic  and nicotinic subtypes due to the unavailability of subtype specific drugs. However, we did confirm the functionality of the homomeric α7nAChR on the D54 and U251 cell lines with the specific agonist PHA 543,613 hydrochloride (Tocris) (data not shown). With this method, we observed an increase in [Ca2+]i upon activation of the AChRs collectively and with selective activation using nicotine and muscarine. These results support the previous findings of our group and those of others, where the expression of several AChRs in GBM cell lines were characterized [2+]i response of ACh in a single GBM cell line. Our time-lapse studies provide an extended examination of AChRs in both GBM cell lines and PDX xenograft lines that recapitulate the heterogeneity of GBMs in situ. The calcium dynamics that we observed with the application of ACh varied between the cell lines (U251/D54) and the PDX xenograft lines (PDX14/PDX22), where a uniform response was observed in the cell lines and the PDX lines had a more diverse response across individual cells. We believe this difference is due in part to the heterogeneity of the cellular profile that PDX xenograft lines retain in culture, compared to the homologous nature of the cell lines. Calcium oscillations were observed under all conditions of AChR stimulation with varying frequency, most likely as a result of IP3-dependent Ca2+ release from internal stores i have been implicated in various cellular changes important in cancer progression including migration, proliferation, and invasion i with AChR activation in GBM cells may not be sustained for long enough to mediate changes in cellular growth.Increases in i with ACh, as bradykinin-mediated GBM migration changes require prolonged Ca2+ oscillations i is essential for MMP-9 activity in both the basal and stimulated conditions, however, future experiments that modulate the transcription and translation of MMP-9 would allow for a clear understanding of the potential influence of AChR activation in these processes.Our findings are supported by a limited number of studies that have found ACh stimulation increases MMP activity and expression in a mouse neuroblastoma cell line and human colon cancer cell line, respectively ,76. Howe2+]i demonstrated that the increase in MMP-9 activity occurs via a Ca2+-dependent mechanism, regardless of the activation status of the AChRs. These results may also suggest that intracellular calcium is required for MMP-9 release. As previously mentioned, MMP release is presumed to occur via calcium-dependent exocytosis, however, evidence in support of this mechanism of release is still lacking. While our results do not provide a definitive confirmation, the significant decrease in the presence of both pro- and mature MMP-9 in the external solution would suggest that the amount of MMP-9 outside the cell is greatly diminished. Thus, these results posit that the release of MMP-9 is being inhibited by the chelation of [Ca2+]i. Future studies with verification by proteolytic analysis could prove critical to address these outstanding questions and whether this finding can be extrapolated to other MMPs.Chelation of [CaAChR stimulation caused a significant increase in GBM cell invasion. As noted previously, this was in stark contrast to our transwell migration results, where no significant increase in migration was observed. We believe that this is due to an absence of ion channel activation, but instead, the enhanced activity of MMP-9, as a specific MMP-9 inhibitor (CAS 1177749-58-4) mitigated this effect. These results affirm the highly interactive relationship between GBM cells and the microenvironment they inhabit. While other examples of the influence of the microenvironment on GBM behavior have been increasingly documented, these results provide novel insights into the role of a neurotransmitter. Moreover, this study and another recent study  have ide3 receptor (CHRM3) correlated with decreased patient survival. Our studies would suggest that this is a result of the enhanced ability of these tumors to invade in comparison to tumors that have lower expression. Again, experimentation is needed to validate this correlation. Clinically, a selective muscarinic M3 receptor antagonist has been FDA approved to treat overactive bladder, however, it appears that this drug does not cross the blood–brain barrier [In light of our discovery of the role that AChRs play in GBM invasion, we questioned whether these in vitro findings would translate to differential expression of these receptors in areas of active infiltration in situ. Utilizing the Ivy GAP dataset, we found a significant upregulation of several AChRs. This upregulation suggests that these receptors play a critical role in the promotion of GBM progression, and potentially via the mechanism we have proposed in this study. However, experimental confirmation is still needed to affirm this correlation, and the question of what is leading to the upregulation of these receptors is yet to be answered. Remarkably, we also found that elevated expression of the muscarinic M barrier ,79. ShouSLC44A2-A4 and CHAT expression in the GBM samples. Elevated choline metabolism is well documented in many cancers, where the transport of choline into the cell is considered the rate-limiting step and is considered a therapeutic target [Autocrine signaling loops are often hijacked by malignant cells, resulting in tumor progression. For example, platelet-derived growth factor, nerve growth factor, and EGF have been implicated in autocrine loops in several malignancies ,81,82,83c target ,85. Cholc target ,15. Agaic target .3 receptor was found to significantly correlate with decreased patient survival. Finally, we found that GBMs express other enzymes required to make ACh, suggesting that these tumors could be capable of synthesizing ACh and thus through autocrine signaling, increase invasiveness into the ECM rich brain environment. Collectively, these findings present a novel signaling axis within GBMs that could be targeted therapeutically to mitigate one of the predominant challenges limiting the effective treatment of these deadly tumors.In conclusion, this study is the first comprehensive characterization of AChRs in a large GBM sample cohort, GBM cell lines, and PDX xenograft lines. Our findings suggest that functional AChRs are expressed on GBM cells and play a role in promoting the invasiveness of these tumors via the increased activity of MMP-9. Additionally, we demonstrated that MMP-9 activity is dependent on intracellular calcium. Expression of several AChRs was found to be significantly upregulated at active zones of GBM infiltration in situ and elevated expression of the muscarinic M


Rhaponticum carthamoides (Willd.) Iljin. is an endemic plant species, which is important in Siberian medicine. It possesses adaptogenic properties and has been used for treatment of overstrain and weakness after illness, physical weakness, and mental weariness. The roots of this species obtained after Agrobacterium rhizogenes transformation are rich in caffeoylquinic acid derivatives known as strong antioxidant compounds. The study makes the first evaluation of the cytotoxic and genotoxic activity of transformed root extract (Rc TR extract) in various human cancer cell lines: leukemia cells (K-562 and CCRF-CEM) and lung adenocarcinoma cells (A549). It was found that Rc TR extract inhibited the cell viability of all tested cell lines in a concentration-dependent manner, and leukemia cell lines were more sensitive to plant extract than A549 lung cancer cell line. Additionally, the Rc TR extract reduced the mitochondrial membrane potential and demonstrated genotoxicity against tested cell lines by increasing mitochondrial DNA lesions in ND1 and ND5 genes and causing nuclear DNA damage in TP53 gene. Our results show that Rc TR extract may effectively treat cancer cells by inducing dysfunction of mitochondria. Additionally, the role of mtDNA may be a promising factor in chemotherapy, and it needs further studies. Rhaponticum carthamoides; it has been found to demonstrate antioxidant activity and is capable of inducing apoptosis in glioma cells by disrupting mitochondrial membrane potential and increasing ROS level, altering Bax/Bcl-2 level, activating p53, caspase-3, or caspase-9, inducing DNA damage or PARP cleavage, and increasing the level of phosphorylated H2A.X [The plant antioxidant compounds have long been known to have beneficial effects on human health; however, recent studies indicate that they may also cause apoptosis and death of cancer cells . The plaRhaponticum carthamoides (Willd.) Iljin (Asteraceae) is an endemic plant species whose roots and rhizomes have been used for many years in traditional Siberian medicine. These raw materials are a component in nutraceutical preparations and diet supplements and are used as adaptogenic and anabolic preparations. R. carthamoides has also been reported to alleviate physical weakness and mental weariness [in vitro cultures, especially transformed root cultures; these are characterized by high metabolite content and biomass production in a short time. Our previous study established transformed roots of R. carthamoides by A4 Agrobacterium rhizogenes transformation and showed that these transformed roots contain caffeoylquinic acids and their derivatives and flavonoid glycosides [O-dicaffeoylquinic acid, and a tentatively identified tricaffeoylquinic acid derivative. The transformed roots of R. carthamoides also demonstrated enhanced production of tricaffeoylquinic acid derivatives compared to the normal roots of soil-grown plants, and they offer an attractive alternative to conventional cultivation and obtainment of the valuable secondary metabolites.eariness . Studieseariness , 7. MostR. carthamoides transformed root extract against human glioma cells, the aim of the present study is estimate its cytotoxic and genotoxic activities in two human leukemia cell lines: myeloid (K-562) and lymphoid (CCRF-CEM) and lung cancer cell line (A549) by evaluating cell viability, mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damages, loss of mitochondrial membrane potential, and alteration of mtDNA copy number.In reference to our earlier studies regarding the cytotoxicity of R. carthamoides were previously obtained by the transformation of A4 Agrobacterium rhizogenes [Transformed roots of izogenes . The estizogenes . The roov/v) aqueous methanol as described in our earlier study [w/w) of the aqueous methanol extract (Rc TR extract) was 19.07%. The roots of soil-grown plants (Rc NR extract) were used as a comparison. The yield of Rc NR extract was 18.87%.The lyophilized plant material (10 g dry weight) was extracted with 80% (μg of streptomycin sulfate per 1 mL of culture media and 10% (v/v) heat-inactivated fetal bovine serum (FBS). All cell lines were grown in a humidified incubator at 37°C and 5% CO2. The cell culture reagents were purchased in Lonza .The following cell lines were used: human lung adenocarcinoma A549  and two human leukemia lines—T lymphoblast CCRF-CEM cells  and chronic myelogenous leukemia K-562 . The cell lines were obtained from the American Type Culture Collection . The A549 cells were cultured in DMEM medium, CCRF-CEM, and K-562 cells in RMPI 1640 medium supplemented with 100 units of potassium penicillin and 100 Rc TR extract or Rc NR extract (0.019-5.0 mg/mL). In brief, A549 cells (1 × 104 cells/well), CCRF-CEM cells (1 × 105 cells/well), and K-562 (1 × 105 cells/well) were seeded in a 96-well plate and cultured overnight in the incubator at 37°C and 5% CO2. The medium was then removed and replaced with the fresh medium supplemented with Rc TR extract or Rc NR extract. The cells were incubated for 24 hours, washed once, and centrifuged (300 × g for five minutes at 22°C) and incubated with 0.5 mg/mL of 3--2,5-diphenyl tetrazolium bromide (MTT) at 37°C. After four hours, the MTT solution was discarded carefully, and the formazan crystals were dissolved in DMSO. Finally, the absorbance was measured for each well at a wavelength of 570 nm with background subtraction at 630 nm using a BioTek Synergy HT Microplate Reader . The relative cell viability was expressed as a percentage relative to the untreated (control) cells defined as 100%. All experiments were performed in triplicate.MTT assay was used to determine cell viability after 24-hour treatment with 4 cells/well (A549) and 1 × 105 cells/well (CCRF-CEM and K-562) in 50 μL culture medium and allowed to adhere overnight. Next, the cells were incubated for 24 hours with Rc TR extract, Rc NR extract (IC50 concentration), or without Rc extract (control cells). Then, the cells were preincubated for 30 minutes with 5 μM JC-1 in the HBSS at 37°C in a CO2 (5%) incubator. Finally, the cells were centrifuged (300 × g for 10 min at 22°C) and washed twice with the HBSS. The fluorescence was measured on a BioTek Synergy HT Microplate Reader  with the filter pairs of 530 nm/590 nm and 485 nm/538 nm. The results are shown as a ratio of fluorescence measured at 530 nm/590 nm to that measured at 485 nm/538 nm (aggregates to monomer fluorescence). The experiments were repeated three times for each cell line.The fluorescent dye JC-1  was used to estimate mitochondrial membrane potential. The cells were seeded into 96-well black plates with transparent bottom (Greiner) at a density of 1 × 10Rc TR extract, Rc NR extract (IC50 concentration), or without Rc extract (control cells). Total genomic DNA  was isolated by the QIAamp DNA Mini Kit  from 2 × 106 cells, according to the manufacturer's instructions. DNA concentrations were determined by the spectrophotometric measurement of absorbance at 260 nm, and the purities were calculated by A260/A280 ratio using a BioTek Synergy HT Microplate Reader . The purified DNA was stored at −20°C until further analysis.The cell samples were incubated for 24 hours with ND1 , ND5 , TP53 (tumor protein p53), and HPRT1 (hypoxanthine phosphoribosyltransferase 1). The primer sequences, SLR-qRT-PCR amplification, and damage calculation were performed according to Bijak et al. [The semi-long-run quantitative RT-PCR (SLR-qRT-PCR) was used to quantify mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage . Long ank et al. . SLR-qRTND1 and ND5, were chosen as mitochondrial target and SLCO2B1 and SERPINA1 genes as nuclear target. The primer sequences, qRT-PCR amplification, and mtDNA copy number calculation were performed according to the study described by Bijak et al. [Quantitative real-time PCR (qRT-PCR) was used to assess the relative number of copies of human mitochondrial DNA (mtDNA) using nuclear DNA (nDNA) content as a standard. Two genes, k et al. . Data wap < 0.05) between the samples .The results are calculated as means ± SD. The normality of the data was determined by the Shapiro-Wilk test, and Levene's test was used to assess the equality of variance. The Kruskal-Wallis test and the one-way analysis of variance (ANOVA) with Tukey's post hoc test were used to determine the significant differences (R. carthamoides (Rc TR extract) for 24 hours. Extract from the roots of soil-grown plants (Rc NT extract) was used for comparison. It was found that both Rc extracts inhibited the cell viability of all tested cell lines in a concentration-dependent manner  were more sensitive to Rc TR extract than A549 lung adenocarcinoma. The Rc TR extract possessed similar cytotoxic activity for both tested leukemia cell lines (K-562 and CCRF-CEM). For these cell lines, 50% of cell viability was achieved after treatment with Rc TR extract at a concentration of 0.313 mg/mL  of extract from transformed roots of t manner , but the13 mg/mL . Twice tR. carthamoides root extracts (TR and NR extracts). It was observed that these plant extracts reduced the mitochondrial membrane potential in all tested cell lines compared to the control cells . The lesion rate in the ND1 region increased after treatment with Rc NR extract and Rc TR extract compared to the control cells (cells untreated with roots extracts): 3.6 lesions per 10 kb DNA (Rc NR extract) and 4.7 lesions per 10 kb DNA (Rc TR extract) in K-562 cells. Furthermore, the Rc TR extract also caused a higher lesion rate in the ND1 region in CCRF-CEM cells compared to the Rc NR extract (Rc NR extract and 5.5 lesions per 10 kb DNA after Rc TR extract (without significant differences). Additionally, an increased level of mtDNA damage was found in the ND5 region only in human leukemia cell lines: 2.9 lesion per 10 kb DNA (Rc NR extract) and 4.0 lesions per 10 kb DNA (Rc TR extract) in K-562 cells and 4.3 lesion per 10 kb DNA (Rc NR extract) and 4.6 lesions per 10 kb DNA (Rc TR extract) in CCRF-CEM cells , but no significant differences were found between the tested extracts , chlorogenic acid (5.12 mg/g DW), and 3,5-O-dicaffeoylquinic acid (3.08 mg/g DW) being the main compounds [It is possible that the caffeoylquinic acid derivatives present in ll lines –19. Ekball lines  suggest ative 5.9 mg/g DW,Rc TR extract also possesses cytotoxic and genotoxic activity in various types of cancer cell line, three human cancer cell lines were tested: K-562 chronic myelogenous leukemia, CCRF-CEM leukemic lymphoblasts, and A549 lung adenocarcinoma. It was found that extract from R. carthamoides transformed root inhibited the cell viability of all tested cell lines in a dose-dependent manner (0.019-5 mg/mL) and that the leukemia cell lines CCRF-CEM and K-562 were more sensitive to Rc TR extract than A549 lung adenocarcinoma. For leukemia cell lines, the IC50 was 0.313 mg/mL, and for lung cancer cells, IC50 = 0.625 mg/mL. Mahbub et al. [Rc TR extract. Additionally, our findings indicate that the Rc TR extract was more cytotoxic to the human leukemia and lung cancer cell lines than human patient-derived grade II-IV glioma cell lines and U87MG human glioma cells [Rc TR extract reduced also the viability of glioma cell lines in a dose-dependent manner, reaching about 50% at a concentration of 0.75 mg/mL or 1.0 mg/mL. The differences observed in cells sensitivity to Rc TR extract treatment may reflect the specificity of the tissue or the degree of malignancy.In order to determine whether the b et al.  reportedβ, and STAT3 which are essential in controlling cellular proliferation, tumor survival, and angiogenesis [Rc TR extract reduced the mitochondrial membrane potential in all tested human cancer cell lines: K-562 chronic myelogenous leukemia, CCRF-CEM leukemic lymphoblasts, and A549 lung adenocarcinoma. It has been previously found that Rc TR extract can disrupt mitochondrial membrane permeability in human glioma cells and can cause a decrease in mitochondrial membrane potential [Rc TR extract, was found earlier to reduce the mitochondrial membrane potential in U937 leukemia cells [In chemotherapy, the induction of apoptosis in cancer cells plays an important role  and is cogenesis . The balogenesis  and inhiia cells .Rc TR extract on mtDNA damage in tested cancer cell lines . Our results show an increase level of mtDNA damage in the ND1 region in all cell lines and in the ND5 region only in the leukemia cell lines after treatment with Rc TR extract compared to control, untreated cells.In cancer cells, mtDNA mutations can be induced by ROS and reactive aldehydes, resulting in changes in encoded proteins needed for mitochondrial functions . The preRc TR extract increased nDNA damage in the TP53 region in both leukemia cell lines: K-562 and CCRF-CEM. No difference in the amount of nDNA damage in the TP53 region was observed between A549 cells treated with Rc TR extract and control. Additionally, no significant difference in the level of nDNA damage in the HPRT1 region was found in all three tested cancer cell lines. Our previous study indicated that 24-hour treatment with Rc TR extract resulted in the induction of DNA double strand breaks in human glioma cells [Rc TR extract, induced DNA damage in K-562 leukemia cells.As the release of mitochondrial ROS can cause damage of nuclear DNA, leading to the promotion of apoptosis and activation of transcription factor , the stuma cells  observedRc TR extract but not significantly. Both increases and decreases in the mtDNA copy number elevate the risk of tumorigenesis [Currently, much interest is directed to mtDNA copy number for assessing the functioning of mitochondria. As mitochondria might play a key role in cancer susceptibility and development, it is reasonable to speculate that mtDNA variations or changes of mtDNA copy number may be closely related to various cancers . As repoigenesis , 31, andigenesis . mtDNA cigenesis . In turnigenesis  suggest igenesis . HoweverR. carthamoides can inhibit cell viability and induce apoptosis in various types of cancer cell . These transformed roots, rich in caffeoylquinic acid derivatives, may be an effective drug in cancer treatment. Additionally, the present study showed that Rc TR extract reduced the mitochondrial membrane potential in all tested human cancer cell lines and possessed genotoxic activity by increasing mtDNA and nDNA damages. Rc TR extract shows potent anticancer activity in various cancer cells by inducing mitochondrial dysfunction, but mechanism of action is not clear yet and it needs further studies.Both the present and earlier findings demonstrate that transformed roots of


The protein profile of a 92-biomarker panel was analyzed on cells treated with 0 or 0.1 µM doxorubicin during 6 or 72 h, under normoxia or hypoxia. Hypoxia decreased viability of HepG2 and SNU499. HepG2 was least and SNU449 most tolerant to doxorubicin treatment. Cytotoxicity of doxorubicin increased over time in HepG2 and Huh7. The combination of doxorubicin + hypoxia affected the cells differently. Normalized protein expression was lower for HepG2 than Huh7 and SNU449. Hierarchical clustering separated HepG2 from Huh7 and SNU449. These three commonly used cell lines have critically different responses to chemotherapy and hypoxia, which was reflected in their different protein expression profile. These different responses suggest that tumors can respond differently to the combination of local chemotherapy and embolization.Hepatocellular carcinoma is often treated with a combination of doxorubicin and embolization, exposing it to high concentrations and hypoxia. Separation of the possible synergistic effect of this combination in vivo is difficult. Here, treatment with doxorubicin, under hypoxia or normoxia in different liver cancer cell lines, was evaluated. Liver cancer cells HepG2, Huh7, and SNU449 were exposed to doxorubicin, hypoxia, or doxorubicin + hypoxia with different duration. Treatment response was evaluated with cell viability, apoptosis, oxidative stress, and summarized with IC Intermediate hepatocellular carcinoma (HCC) is treated with image-guided transcatheter tumor therapy by locally infusing a chemotherapeutic agent in a drug delivery system, so called transarterial chemoembolization (TACE) ,2,3,4. TTumor hypoxia is a key hallmark of all solid tumors, including HCC. An inevitable consequence of chemoembolization therapy is the de novo formation of hypoxic regions within the tumor . HypoxicHuman cancer-derived cell lines are the most widely used in vitro models to study cancer mechanistically and to validate the efficacy of cancer treatments . One of In this study, the objective was to investigate the tolerance to DOX and/or hypoxic treatment of three commonly used liver cancer cell lines ,26,27. H2 was confirmed by measurement of HIF1α and PDK1 after CoCl2 treatment confirmed presence of hypoxia  of HepG2 cells to DOX decreased 1600-fold from 6 to 72 h of exposure in normoxic conditions . These cell lines were selected based on previously published data on their response to DOX and their morphological features.50 values for HepG2 and SNU449. Huh7 and HepG2 are epithelial cell lines, characterized by a high expression of E-cadherin and low expression of Vimentin [50 values of DOX from different studies vary vastly. For example, after 48 h DOX exposure on HepG2 cells IC50 values varied from 0.038 to 1.9 µM [50 values for DOX in human carcinoma cell lines decrease with exposure time [50 is reported for HepG2 cells when increasing exposure time from 6 to 48 h, and similar effects (30-fold decrease) are observed in a ovarian cancer cell line when increasing exposure time from 2 to 12 h. [50 decreased between 60- and 500-fold, depending on cell line and increase in exposure time. This suggests that our cell lines were highly responsive to DOX. Interestingly, the SNU449 cell line showed higher tolerance at 72 h than after 48 h, having possibly built up resistance to DOX treatment. Several mechanisms have been described to contribute to the acquired resistance to DOX [The observed tolerance to DOX was significantly different between the cell lines, where HepG2 cells exhibited the lowest and SNU449 cells the highest tolerance. For example, at 72 h exposure to DOX there was a 580-fold difference between ICVimentin . In contVimentin ,31. The Vimentin ,31. In sVimentin . HoweverVimentin ,34 up toVimentin ,35. Alsoo 1.9 µM ,37. Thise to DOX . Firstlye to DOX . Unfortue to DOX ,41. Thise to DOX . In our e to DOX , which ce to DOX . An in vThe difference in viability of cells after treatment with DOX is also reflected in their different expression of the apoptotic marker Annexin V. Under normoxia, Annexin-V increased in the Huh7 and SNU449 cells treated with 0.1 and 1 µM DOX for 24 h. This is in line with our viability data, which show a decrease of 3% in cell viability of Huh7 cells after 24 h of exposure to 0.1 µM and 29% after 24 h of exposure to 1 µM DOX in normoxic conditions. In contrast, the SNU449 cells had no increased levels of Annexin V, after treatment with DOX in hypoxic conditions. This is in line with our viability data, which show that SNU449 cells experience no decrease in cell viability at 0.1 µM and 1 µM DOX. Under hypoxia, Huh7 cells showed a significant increase of Annexin V, after 24 h exposure to 0.1 µM and 1 µM DOX. This partially corresponds to our viability data, which show no decrease in cell viability of Huh7 cells after 24 h of exposure to 0.1 µM, but a 15% after 24 h of exposure to 1 µM DOX in hypoxic conditions. Remarkably, HepG2 cells showed no change in the apoptotic marker Annexin V, suggesting other mechanism may be responsible for the decreased viability. Despite the widespread clinical use of DOX, its antiproliferative and death-inducing signal cascades are not yet fully understood. Studies have suggested that decreased viability seen after DOX treatment is a result of apoptosis, necrosis, cell cycle arrest, and senescence ,46,47, w2 resulted in varying responses of the three cell lines. HepG2 and SNU449 cell viability declined when exposed to hypoxia. Huh7 had unaffected or slightly increased cell viability in hypoxic conditions, suggesting that Huh7 has a higher tolerance to hypoxia. It is important to note that HIF1α activity varies between different cancer cell lines under the same level of hypoxia [2-induced hypoxia, but recovered to normal levels at 72 h. This suggests that these cells adapted to the hypoxic conditions over time. In contrast, Huh7 has a higher tolerance to hypoxia, but also lower baseline levels of stabilized HIF1α and PDK1 in normoxic conditions. In agreement to our findings, a study on six human breast cancer cell lines found high basal levels of VEGF A and low HIF-1α and HIF-2α induction was correlated with improved survival under hypoxia [2, and then activates transcription of both VEGF A and CAIX [Inducing hypoxia with CoCl hypoxia . The difand CAIX . In our and CAIX ,50 was dTolerance of cancer cell lines to chemotherapeutic agents can increase or decrease in hypoxic conditions. Increased tolerance to chemotherapeutics under hypoxic conditions are observed in human embryonal carcinoma testicular germ cell tumor cell lines , HCC cel2 does not create an actual oxygen deprivation, but mimics hypoxia by binding to prolyl hydroxylase containing domain proteins that activate HIF-1α, there is still an abundance of reactive oxygen species, which might have contributed to the DOX cytotoxicity [2. A low exposure of 0.1 µM DOX induced oxidative stress in all cell lines after 24 h, which is in line with previous reports [2-induced hypoxia was combined with DOX treatment. This suggests that oxidative stress did not contribute to DOX cytotoxicity in the SNU499 cell line. Elevation of intracellular mitochondrial ROS levels in tumors is known to activate PKD1 and NF-κB, leading to upregulation antiapoptotic proteins such as A20 and cIAPs [It has been hypothesized that cells that proliferate less in hypoxia will be more tolerant to chemotherapeutic agents, as the chemotherapeutic agents act on dividing cells . DOX hastoxicity . In linetoxicity , we foun reports . A highend cIAPs . Therefond cIAPs .2 [2. In our study, we found different levels of HIF1α and PDK1 in the three cell lines under the same oxygen conditions, which could explain differences in their adaptation to hypoxia and their sensitivity to DOX. In addition, there were differences in generation of ROS between the different cell lines under normoxia and hypoxia, before and after DOX treatment, which would further contribute to the cytotoxic effect of both hypoxia and DOX.DOX is known to upregulate and activate HIF-1α, and HIF-1α targeting strategies have been suggested to enhance the effect of DOX treatment . Possibl2 . It is t2 is a hypoxic mimetic that functions by stabilizing HIF-1α. It has been shown that the difference in oxygen levels during hypoxia could affect cell behavior, where induction of apoptosis was observed when oxygen levels decreased below 0.5%, but not at oxygen levels of 1–3% [Another possible explanation of the different responses between cell lines, both in historical data and our results, could be the difference in how hypoxia was created. Chemical hypoxia with CoCl of 1–3% . Interes of 1–3% , which iA final important finding in our study was the difference in oncogenic protein profile between cell lines. Previous studies have shown a high similarity in gene expression between Huh7 and HepG2 cell lines and HCC tumor tissue, based upon rank-based gene expression ,27. The 2, Sigma-Aldrich, Darmstadt, Germany) stock solutions were prepared by dissolving CoCl2 in sterile water (25 mM) and filtering through a syringe filter (0.22 µm).Doxorubicin hydrochloride (DOX HCl) was purchased from Toronto Research Chemicals, Canada. DOX stock solutions (100 mM) were prepared by dissolving the DOX in DMSO . RIPA buffer, resazurin sodium salt and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich . Dulbecco’s Modified Eagle’s Medium (DMEM), Roswell Park Memorial Institute medium (RPMI), fetal bovine serum (FBS), and trypsin-EDTA were purchased from Gibco. Cobalt (iii) chloride hexahydrate (CoClFBS) and 1% antibiotic–antimycotic solution . Cells were cultured at 37 °C with 5% CO2 under standard cell culturing conditions  before making the 0, 0.01–1000 µM DOX concentration range in CCMFBS + CoCl2  + DOX was added to each well. Plates were again incubated in a humidified incubator with 5% CO2 at 37°C for the desired exposure time , with and without chemically induced hypoxia  or hypoxic conditions  for 6, 24, and 72 h. HIF1α and PDK1 expression was measured using Hypoxic Response Human In-Cell ELISA Panel  according to manufacturer’s guidelines. Plates were scanned using an Odyssey imager . Expression values were obtained by subtracting background values from wells with negative controls for each condition and normalized to the corresponding Janus Green fluorescence values for each well.Cells were seeded into 113851) in a microplate format and by flow cytometry. DCFDA is fluorogenic dye that measures ROS activity within the cell. After diffusion in to the cell, DCFDA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into DCF, a highly fluorescent compound with maximum excitation and emission spectra of 495 nm and 529 nm respectively. Cells were seeded into 96-well plates with clear bottom and black sides at a density of 2.4 × 104 cells/well for the microplate assay, and into 24-well plates at 1.2 × 105 cells/well for flow cytometry and left to adhere overnight. On the next day, cells were stained with 25 µM DCFDA for 45 min at 37 °C, according to manufacturer’s guidelines. Following this step, cells were exposed to 0, 0.1 µM and 1 µM DOX in normoxic (CCMFBS) or hypoxic conditions . After 6 h of treatment, fluorescence was measured at 485 nm excitation and 535 nm emission wavelengths using a Fluostar Omega plate reader and BD FACSCallibur. Results of the microplate assay are shown as fold change fluorescence. Results of the flow cytometry measurements are shown as percentage of mean DCF signal intensity of control conditions.Oxidative stress was measured using DCFDA—Cellular Reactive Oxygen Species (ROS) Detection Assay Kit  or hypoxic conditions  for 24 h, following which apoptosis was assessed by flow cytometry using eBioscience™ Annexin V Apoptosis Detection Kit FITC (88-8005-72) according to manufacturer’s guidelines. Briefly, cells were washed with 1× Binding Buffer and incubated with a 1/20 dilution of fluorochrome-conjugated Annexin V for 10 min, after which fluorescence was measured at 485 nm excitation and 535 nm emission wavelengths using BD FACSCallibur. Results are shown as percentage of mean Annexin V signal intensity of normoxic untreated condition.Cells were seeded into 24-well plates at 1.2 × 106-well plates at 2 × 105 per well and left to adhere overnight. DOX stock solution was diluted to 0.1 µM in CCMFBS (+ CoCl2). Eight different conditions were tested per cell line: normoxia or chemically induced hypoxia with exposure to 0 or 0.1 µM DOX during 6 or 72 h, with three replicates per experimental condition. Medium and cell debris were removed and remaining cells washed with PBS. Cells were detached with 100 µL ice-cold RIPA containing protease inhibitors (Sigma Aldrich), and cells were scraped off and aspired :50% inhibitory concentration (IC50) of DOX. DOX IC50 values were determined per cell line, for each exposure time, for both normoxic and hypoxic conditions. Differences between DOX IC50 with and without hypoxia per cell line was tested statistically with a 2-way ANOVA and Tukey’s multiple comparisons test (significant when adjusted p < 0.05) in GraphPad Prism. The effect of hypoxia was assessed by taking the ratio of cell viability under hypoxic and normoxic conditions. The effect of hypoxia on cell viability was tested statistically with a one-sample t-test (difference from 1) in GraphPad Prism.The blank wells were empty in the plate while control wells contained cells in medium without any additional DOX treatment. Cell viability data vs. DOX concentrations were plotted in GraphPad Prism  and fitted with the “inhibitor vs. response—variable slope analysis” to determine the Protein expression data from all different treatments on the three cell lines C was obt2, Huh7, and SNU449) have critically different responses to chemotherapy and hypoxia. This was also reflected in their oncogenic protein profile and their response to hypoxia and oxidative stress. A synergic effect of hypoxia and treatment with DOX was only observed in HepG2 cells, while Huh7 and SNU449 might have developed escape mechanisms from both treatment and hypoxia. These escape mechanisms are of uttermost importance when studying chemotherapeutic agents. Adaptation of tumor cells to hypoxia is believed to be the main driver for selection of more invasive and therapy-resistant cancer phenotypes [In this study, we present our findings that three established and commonly used liver cancer cell lines (HepGenotypes . Our resenotypes .


The CDK7 inhibitors (CDK7i) ICEC0942 and THZ1, are promising new cancer therapeutics. Resistance to targeted drugs frequently compromises cancer treatment. We sought to identify mechanisms by which cancer cells may become resistant to CDK7i. Resistant lines were established through continuous drug selection. ABC-transporter copy number, expression and activity were examined using real-time PCR, immunoblotting and flow cytometry. Drug responses were measured using growth assays. ABCB1 was upregulated in ICEC0942-resistant cells and there was cross-resistance to THZ1. THZ1-resistant cells upregulated ABCG2 but remained sensitive to ICEC0942. Drug resistance in both cell lines was reversible upon inhibition of ABC-transporters. CDK7i response was altered in adriamycin- and mitoxantrone-resistant cell lines demonstrating ABC-transporter upregulation. ABCB1 expression correlated with ICEC0942 and THZ1 response, and ABCG2 expression with THZ2 response, in a panel of cancer cell lines. We have identified ABCB1 upregulation as a common mechanism of resistance to ICEC0942 and THZ1, and confirmed that ABCG2 upregulation is a mechanism of resistance to THZ1. The identification of potential mechanisms of CDK7i resistance and differences in susceptibility of ICEC0942 and THZ1 to ABC-transporters, may help guide their future clinical use. Cyclin dependent kinase CDK7), along with cyclin H and the accessory protein MAT1, forms the CDK-activating kinase (CAK), which functions to activate CDKs by T-loop phosphorylation . CAK is , along wCell cycle deregulation is a common feature of cancer and a number of cell cycle CDK inhibitors have been approved as cancer therapeutics, most prominently CDK4/6 inhibitors, exemplified by palbociclib, for the treatment of advanced oestrogen receptor positive (ER+) breast cancer . By contThe emergence of resistance to cancer treatment remains a major problem in cancer therapy. Cancer cells can adapt and become resistant to drugs in a multitude of ways, including, but not limited to, mutations that disrupt drug-target engagement, activating mutations in drug targets and downstream pathways, changes in gene expression levels of targets, altered drug metabolism and alterations in cell death pathways . In addiWhilst CDK7 inhibitors (CDK7i) represent a promising therapeutic approach, development of resistance in the clinic is possible, therefore identifying potential resistance mechanisms preemptively is of great importance. We have developed models of acquired resistance to both ICEC0942 and THZ1 in the breast cancer cell line MCF7, identifying upregulation of ABC-transporters as mediators of resistance. These findings may be informative for future patient selection strategies and combination treatment regimes.50 became growth-arrested after 1–2 weeks, so could not be continually passaged. Cells treated with 800 nM ICEC0942 remained growth arrested until resistance developed after 4 months, eventually giving rise to the ICEC0942-resistant line (MCF7-942R).To investigate mechanisms of acquired resistance to the CDK7i ICEC0942 and THZ1, we generated MCF7-derived drug resistant cell lines through prolonged culturing in the presence of ICEC0942 or THZ1. THZ1 resistance was achieved by initially exposing the cells to 50 nM THZ1, approximately the concentration required for 50% growth inhibition. This was increased gradually up to a concentration of 250 nM, with passaging once a week, until the cells became stably THZ1-resistant after 3 months. A different strategy was used to achieve ICEC0942 resistance, as even cells exposed to drug concentrations well below ICR showed a 29-fold resistance to ICEC0942  showed a 13-fold resistance to THZ1 .THZ1 inhibits CDK7 by covalently targeting a cysteine residue (C312), which lies outside the ATP-binding domain , whereasR cells, ABCG2 expression was increased 140-fold, relative to MCF7 cells , ABCG2 (HEK293ABCG2) or empty vector (HEK293)  and ABCG2 (P < 0.0001) and THZ2 response  [R) [R    and a miR) [R) , 29. qRT [R Fig.  and flow [R Fig. . NCI-ADRµM) Fig. , whereasely Fig. .Fig. 6ChR was confirmed to have high expression and pump activity of ABCG2, by qRT-PCR  as frozen stocks. The NCI Adriamycin-resistant cell line (NCI-ADRR) was provided by Dr. Ernesto Yagüe, and OVCAR8 by Dr. Paula Cunnea, both from Imperial College London . The MCF7 mitoxantrone-resistant cell line (MCF7-MXR) was provided by Dr. E Schneider from the University of Maryland . HEK293 cells, stably transfected with either empty pcDNA 3.1 vector (HEK293), pcDNA3.1 containing ABCB1 (HEK293ABCB1) or ABCG2 (HEK293ABCG2) have been described [R, which were maintained in phenol-red free RPMI, supplemented with 10% FCS and 1 nM estradiol. Cell lines were cultured up to a maximum of 20 passages beyond initial thawing. Cell cultures were routinely tested for Mycoplasma infection by assay of culture supernatants, and found to be negative, using the MycoAlert Mycoplasma Detection Kit .MCF7 cells were obtained from the ATCC (escribed . All celR) was established by growing MCF7 cells in the continued presence of ICEC0942 at 800 nM for 4 months, splitting when cells neared confluence. The MCF7 THZ1-resistant cell line (MCF7-THZ1R) was established by initially growing MCF7 with THZ1 at 50 nM. Cells were split when they neared confluence and the THZ1 concentration then raised by 25 nM increments, up to a maximum of 250 nM, over a 3-month period. The independent MCF7 ICEC0942-resistant cell line (ICR-MCF7-942R) was established by growing MCF7 cells in the continued presence of ICEC0942 at 400 nM for 3 months, splitting when cells neared confluence. After drug resistance was established, the cell lines were maintained in culture with 800 nM ICEC0942 (MCF7-942R), 400 nM ICEC0942 (ICR-MCF7-942R), or 250 nM THZ1 (MCF7-THZ1R), with medium and drug changes every 3–4 days.The MCF7 ICEC0942-resistant cell line (MCF7-942ICEC0942 synthesis has been described  and was 50) were calculated from log dose-response curves in GraphPad prism v7.0. All dose-response curves are presented with error bars for standard errors of the mean (SEM) of six technical replicates. A minimum of three independent experiments were conducted and the average fold resistance of the drug resistant cell lines, MCF7-942R and MCF7-THZ1R, relative to the parental cell line, MCF7, was determined thus; fold resistance = GI50 resistant cell line/GI50 parental cell line [The sulphorhodamine B (SRB) assay was used to assess drug sensitivity, as previously described . Drug coell line . For ABCWhole cell lysates were prepared in RIPA buffer (Sigma-Aldrich), supplemented with protease and phosphatase inhibitors , and immunoblotting was carried out as previously described . AntibodR and NCI-ADRR. Cells were trypsinised and resuspended in phenol red-free DMEM at a concentration of 1 × 106 cells per ml. Twenty microliters of the EFFLUXX-ID Green Detection Reagent (in DMSO) was diluted in 1 ml DMEM (phenol red-free). Cells (2 × 106) were incubated with 50 µl of the diluted EFFLUXX-ID Green Detection Reagent and the fluorescence intensity of each sample was measured for 10,000 events every 5 min for 30 min, on a BD LSR II Flow Cytometer . The same procedure was used to assess the pump efflux activity of ABCG2 in, MCF7, MCF7-THZ1R and MCF7-MXR, except Hoechst 33342 [The EFLUXX-ID Green Multidrug Resistance Assay Kit  was used to assess the pump efflux activity of ABCB1 in MCF7, MCF7-942st 33342  was usedR cells was carried out by reverse transfection, using Lipofectamine RNAiMAX transfection reagent (Thermo Scientific), with ABCG2-specific siRNA  or non-targeting siRNA, (Dharmacon ON-TARGETplus Nontargeting Pool) in either six well plates , or 96 well plates , at a final concentration of 25 nM. ABCB1 knockdown in MCF7-942R cells was carried out in the same way but with ABCB1-specific siRNA (Dharmacon SMARTpool ON-TARGETplus) or non-targeting siRNA (Dharmacon ON-TARGETplus Nontargeting Pool). RNA preparation, qRT-PCR, protein preparation, immunoblotting and drug sensitivity assays were then carried out as described above. Primers used in sequencing and qRT-PCR are listed in Supplementary Table Knockdown of ABCG2 in MCF7-THZ1t-test. The Pearson correlation coefficient was used for gene expression analyses. To assess whether ABC-transport inhibitors significantly alter the GI50 of CDK7i, in CDK7 inhibitor-resistant cell lines, the extra sum-of-squares F test was used.Statistical analyses were carried out in Graphpad prism v7.0. Pairwise comparisons were performed using the Student Supplementary Methods.Supplementary Figure Legends.Supplementary Figures.


Class I histone deacetylases (HDACs) generally promote cell proliferation and tumorigenesis, whereas class IIA HDACs like HDAC4 and HDAC5 may promote or impede cancer development in a tissue-dependent manner. In urothelial carcinoma (UC), HDAC5 is often downregulated. Accordingly, HDAC5 was weakly expressed in UC cell lines suggesting a possible tumor-suppressive function. We therefore characterized the effects of stable HDAC5 expression in four UC cell lines  with different phenotypes reflecting the heterogeneity of UC, by assessing proliferation, clonogenicity and migration ability. Further, we detailed changes in the proteome and transcriptome by immunoblotting, mass spectrometry and RNA sequencing analysis. We observed that HDAC5 overexpression in general decreased cell proliferation, but in one cell line (VM-Cub-1) induced a dramatic change from an epitheloid to a mesenchymal phenotype, i.e., epithelial-mesenchymal transition (EMT). These phenotypical changes were confirmed by comprehensive proteomics and transcriptomics analyses. In contrast to HDAC5, overexpression of HDAC4 exerted only weak effects on cell proliferation and phenotypes. We conclude that overexpression of HDAC5 may generally decrease proliferation in UC, but, intriguingly, may induce EMT on its own in certain circumstances. Histone deacetylases (HDACs) remove acetyl groups from histone or non-histone proteins and regulate gene expression as components of multiprotein repressor complexes ,2,3,4. IOur group studies HDAC function and the therapeutic potential of HDAC inhibitors in urothelial carcinoma (UC), the most common type of urinary bladder cancer. In accord with observations in many other cancers, we have previously observed that experimental downregulation or pharmacological inhibition of class I HDACs, in particular of HDAC1 and HDAC2 combined, strongly impairs proliferation and survival of UC cells . The staIn a previous investigation of HDAC4 expression in UC cell lines and published microarray data sets, we found variable levels of HDAC4 protein and mRNA, respectively, in UC cell lines and tissues . OverexpIn accord with the data from the large TCGA study, we observed that HDAC5 was consistently lower expressed in UC cell lines than in normal urothelial cells. Taken together, these findings suggest a possible tumor-suppressive function of HDAC5 in UC. We therefore characterized in detail the effects of stable expression of HDAC5 on four urothelial carcinoma cell lines with different phenotypes, which reflect the heterogeneity of the disease. We observed that HDAC5 overexpression in general decreased cell proliferation, but in one cell line induced a dramatic change from an epitheloid to a mesenchymal phenotype, i.e., epithelial-mesenchymal transition (EMT). These phenotypical changes were further followed up by comprehensive proteomics and transcriptomics analyses. While HDAC5 has previously been identified as a factor in fibrosis and cancer progression, this is, to our knowledge, the first report demonstrating that HDAC5 on its own may induce EMT in certain circumstances. In accord with our previous report, overexpression of HDAC4, in comparison, had only weak effects on UC cells.To determine the endogenous protein levels of class II HDACs, HDAC4, HDAC5, and HDAC7, protein lysates from ten different UC cell lines (UCCs) and two non-cancerous controls, namely the benign urothelial cell line HBLAK and the primary urothelial culture UP245, were subjected to immunoblotting. Expression of HDAC4 and HDAC7 was variable among the cell lines. For instance, SW1710 and 5637 cells had stronger HDAC4 expression compared to the low levels in HT1376, UM-UC-3, 253J, J82, 639V and HBLAK. The expression of HDAC4 was nearly undetectable in RT112, VM-Cub-1, BFTC905, and UP245 cells. HDAC7 expression was also variable, being clearly stronger in 5637, UM-UC-3, 253J, as well as the controls HBLAK and UP245, compared to other UCCs. Notably, HDAC7 regularly appeared as a double band, which could correspond to several isoforms known to be generated by alternative splicing. In contrast to HDAC4 and HDAC7, HDAC5 levels in UCCs were uniformly low and often below the detection limit. The highest expression was observed in the normal UP245 culture A.Due to its low expression, we were primarily interested in the effects of HDAC5 overexpression in UCCs. Therefore, lentiviral-based vectors were used to introduce HDAC5 into four UCCs; paired control cell lines were generated using vector only. The parental four cell lines used, RT112, VM-Cub-1, SW1710 and UM-UC-3, differ in their phenotypes with decreasing epithelial and increasing mesenchymal morphology in that order. Stable overexpression of HDAC5 was achieved in all four cell lines B.We next measured cell proliferation by MTT assay at different time points over 96 h. Until 72 h (48 h in the case of UM-UC-3), HDAC5 and vector-only transduced cell lines grew quite similarly, but at 96 h proliferation of HDAC5-expressing RT112, SW1710 and UM-UC-3 cells was clearly diminished  in comparison to their respective vector-only cells . InteresThe decrease in proliferative ability over time conferred by HDAC5 was also reflected in clone formation assays. The ability to form clones following seeding at low density in tissue culture plates was strongly diminished in HDAC5-transduced RT112, SW1710 as well as UM-UC-3 cells, and to a lesser extent in VM-Cub-1, compared to their respective vector-only controls . Upon seAmong UCCs, almost exclusively, cell lines with a more mesenchymal morphology form colonies in soft agar. Accordingly, the morphology of HDAC5-transduced VM-Cub-1 cells changed towards a more mesenchymal morphology and the cells grew in a more dispersed pattern rather than as tight colonies a. We therefore investigated markers of epithelial-mesenchymal transition by immunoblotting. Indeed, in VM-Cub-1 HDAC5-transduced cells, the amounts of the epithelial markers Cytokeratin 5 and E-Cadherin were diminished compared to the control, and the expression of the mesenchymal marker Vimentin was increased to a similar level as in SW1710 and UM-UC-3 cells b. In theSince a more mesenchymal phenotype is often associated with increased migratory ability, we compared HDAC5-transduced to vector-only transduced UCCs in cell migration assays. A clear increase in migration was seen for HDAC5-expressing VM-Cub-1 cells over the entire duration of the experiment, whereas no significant difference in migration velocity was observed among vector-only and HDAC5-transduced SW1710 cells. RT112 and UM-UC-3 cells appeared to migrate slightly faster at earlier time points, but the differences were not statistically significant c. To characterize the overall changes in the proteome of the UCCs following HDAC5 overexpression, we performed high-throughput proteomics analysis by mass spectrometry. In accord with the morphological changes, by far the most profound alteration of the proteome was observed in VM-Cub1 cells where more than half of the quantified proteins (1747 proteins) were significantly differently abundant in HDAC5-transduced versus vector-only cells. In comparison, a moderate number of significant changes were observed in SW1710 (145 proteins), and UM-UC-3 cells (154 proteins), and comparatively few in RT112 (33 proteins) . HierarcFinally, we searched for pathways that might underlie the different reaction of VM-Cub-1 to HDAC5 overexpression compared to the other cell lines. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis identified metabolic processes, especially “amino sugar and nucleotide sugar metabolism”, as well as “proteasome” and “ribosome” as consistently enriched in VM-Cub-1 compared to the other three UC cell lines . The chap ≤ 0.05 and at least two-fold differences. As in the proteome analysis, more moderate numbers of genes were differentially expressed in SW1710 and UM-UC-3, namely 1,265 and 733 genes, respectively. Likewise, the lowest number of changes was observed in RT112, with 261 genes differentially expressed  to an aggressive, undifferentiated and mesenchymal cell line (UM-UC-3). The most interesting case was however the VM-Cub-1 cell line, which is considered as representative of the basal subtype and moderately differentiated. This cell line was not significantly impeded in its growth, but underwent a dramatic phenotypic change with obvious EMT and acquisition of the ability for anchorage-independent growth. The different response to HDAC5 between VM-Cub-1 cells and the other cell lines was not only visible morphologically, but was also reflected in the proteome and transcriptome of this cell line. Compared to the other cell lines, changes in VM-Cub-1 were more numerous and more pronounced. It is not clear why VM-Cub-1 reacts differently from the other UCCs to HDAC5. Evidently, SW1710 and particularly UM-UC-3 already possess mesenchymal morphology in the absence of HDAC5. RT112, on the other hand, is more strictly epithelial and grows in tighter clusters than VM-Cub-1. Indeed, we have observed in a previous study that VM-Cub-1 cells but not 5637, another strictly epithelial cell line, underwent EMT in response to overexpression of the lncRNA HOTAIR . InteresIt is indeed intriguing that expression of HDAC5 on its own is capable of inducing EMT in certain circumstances, and notably, appears to be more effective than even TGFβ, which can promote enhanced migration and invasion in some UCCs . While tObviously, overexpression of any protein in a cell line has the potential to cause unspecific effects irrelevant to its normal biological function. This is also a concern in our study. However, the comparison between the effects of HDAC4, which were modest at most, and those of HDAC5, which were prominent, indicates a high degree of specificity and suggests that the effects observed with HDAC5 overexpression reflect biologically relevant actions.In conclusion, according to global gene expression analyses, the class IIA HDAC4 and HDAC5 are both lower expressed in urothelial carcinoma than in normal bladder tissue. In UC cell lines, however, HDAC5, but not HDAC4 is consistently weakly expressed. Accordingly, lentiviral transduction of HDAC5, but not HDAC4 had significant effects on cellular properties. Supporting a tumor-suppressive function of HDAC5 in UC, its overexpression diminished growth in three out of four cell lines. However, induction of EMT in a fourth cell line hints at a potential of HDAC5 to promote a more aggressive phenotype in individual cases. Strikingly, we found HDAC5 overexpression on its own can be capable of inducing EMT. Our findings call for further analyses of the relation between HDAC5 on the one hand, and EMT and fibrosis, on the other hand, in cancer and other diseases.Finally, the overall aim of our project is defining an optimal HDAC target profile for HDAC inhibitors for the treatment of UC . In this2. As a benign urothelial control, we used the HBLAK cell line  VM-Cub1, RT112, SW1710, and UM-UC-3 were provided by Dr. M. A. Knowles , Dr. J. Fogh  and Dr. B. Grossmann  or by the DSMZ . They were cultured in DMEM GlutaMAX-I  supplemented with 10% fetal calf serum  at 37 °C in a humidified atmosphere of 5% COll line  and TNFα ) were used at 5 ng/mL and 20 ng/mL, respectively.The plasmid pcDNA3.1+HDAC5-FLAG was a kind gift from Eric Verdin (Addgene plasmid #13822) . BrieflyColony forming potency of vector and HDAC5 expressing UCCs was assessed by Giemsa staining. One thousand cells (in triplicates) were seeded in 6 well plates and cultured for 8 d or 10 d, for RT112, until macroscopically visible colonies had developed. Then, colonies were washed with PBS, fixed in methanol and stained with Giemsa .Soft agar assays were performed by a standard method  in DMEM Cell viability as an indicator of proliferation was determined by MTT -2,5-diphenyltetrazolium bromide, Sigma-Aldrich) assays as described . One tho4 cells/well) were seeded around Ibidi inserts in 50 µl medium on either side. The next day, inserts were detached to form a gap between the two sections. Cells were once gently washed with media and left to migrate for up to 24 h. Images were taken at the indicated time points and cell migration was measured as the distance between opposite cell fronts and plotted. Measurements were performed in triplicates. Vector-only and HDAC5-transduced UCCs  and MACSQuantify software as previously described .Immunoblot analysis of whole cell extracts was performed as described . Cells wCells were harvested with Qiazol  and lysates were stored at −80 °C. Total RNA was then isolated by the Qiagen RNeasy Mini Kit . RNA quality was checked by spectrophotometry. w/v) 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate, 30 mM Tris-HCl, pH 8.0) and prepared for mass spectrometric analysis, as described elsewhere [To study the effect of HDAC5 overexpression on selected cell lines, quadruplicates from individual culture dishes were prepared from RT112, VM-Cub-1, SW1710, and UM-UC-3 cells expressing HDAC5 as well as corresponding and HBLAK vector-only cells. Cells were harvested and protein lysates prepared in an aqueous urea containing buffer  was used with standard parameters. Spectra were matched against sequence data from the Homo sapiens proteome . Further search parameters and parameters for peptide and protein acceptance and quantification were essentially as described previously .Further analysis of quantitative data was carried out with Perseus  within the R environment . Here, only proteins showing at least two different peptides were considered and proteins showing at least three valid quantitative values in at least one group in the respective comparison. For the identification of HDAC5 affected proteins and differences between the cell lines, a two way ANOVA followed by a Benjamini Hochberg correction and by Tukey’s honest significance tests was carried out on log2 label-free quantification intensities after missing values were filled in with random values from a normal distribution . Additionally, Student’s t-test were calculated for pairwise comparisons of HDAC5-transduced and vector-only cells and cutoffs were determined by the significance analysis of microarrays method . Differences of the mean values of log2 label-free quantification intensities were further used for categorical annotation enrichment analyses.Total RNA samples used for transcriptome analyses were quantified  and quality measured by capillary electrophoresis using the Fragment Analyzer and the Total RNA Standard Sensitivity Assay . All samples in this study showed high quality RNA Quality Numbers . The library preparation was performed according to the manufacturer’s protocol using the ‘TruSeq Stranded mRNA Library Prep Kit’ from Illumina®. Briefly, 250 ng total RNA were used for mRNA capturing, fragmentation, the synthesis of cDNA, adapter ligation and library amplification. Bead purified libraries were normalized and finally sequenced on the HiSeq 3000/4000 system  with a read setup of 1x150 bp. The bcl2fastq tool was used to convert the bcl files to fastq files as well for adapter trimming and demultiplexing. Data analyses on fastq files were conducted with CLC Genomics Workbench . The reads of all probes were adapter trimmed (Illumina TruSeq) and quality trimmed . Mapping was done against the Homo sapiens (hg38)  genome sequence. After grouping of samples  according to their respective experimental condition, multi-group comparisons were made and statistically determined using the Empirical Analysis of DGE . The Resulting P values were corrected for multiple testing by FDR and Bonferroni-correction. A P value of ≤0.05 was considered significant.RNA-Seq data were further analyzed with the Ingenuity Pathway analysis software  using the expression data with p(FDR) ≤ 0.05 and fold-change ≥ 1.7. The number of gene hits with this filter were 606 (RT112), 8000 (VM-Cub-1), 2419 (SW1710), and 1527 (UM-UC-3) in HDAC5-expressing cells compared to their vectors.p < 0.05 was considered as statistically significant.Data are represented as the mean with standard deviation (SD). Statistically significant differences between two groups were analyzed using the unpaired Student’s t-test with GraphPad. A minimum p value of