Byrsonima crassifolia (L.) Kunth and B. verbascifolia (L.) DC.) and tapereba (Spondias mombin) are Amazonian fruits that contain bioactive compounds. Biochemical and molecular characterization of these fruits can reveal their potential use in preventing diseases, including cancer. The extracts were characterized regarding the presence and profile of carotenoids by high-performance liquid chromatography (HPLC), total phenolic content by the Folin–Ciocalteu assay, and antioxidant activity by antioxidant value 2,2-diphenyl-1-picrylhydrazyl (DPPH) content analysis, 22,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) content analysis, Ferric-Reducing Ability of Plasma (FRAP), and Oxygen Radical Absorbance Capacity (ORAC) analysis. The extracts of tapereba and murici studied were important sources of total carotenoids and lutein, respectively. The extracts were then tested for their effect on the viability of the A2780 ovarian cancer (OC) cell line and its cisplatin (CDDP)-resistant derived cell line, called ACRP, by using MTT -2,5-diphenyltetrazolium bromide) assays. Their influence on cell cycle and apoptosis were analyzed by using flow cytometry. Murici and tapereba cell extracts exhibited a strong bioactivity by inhibiting A2780 and ACRP cell viability by 76.37% and 78.37%, respectively, besides modulating the cell cycle and inducing apoptotic cell death. Our results open new perspectives for the development of innovative therapeutic strategies using these Amazon fruit extracts to sensitize ovarian cancer cells to current chemotherapeutic options.Murici ( Ovarian carcinoma (OC) is a highly heterogeneous disease and despite advances in understanding it, patients suffering from this disease still have poor prognostic rates related to late diagnosis and rapid progression . In addiAlternative approaches or combination therapies including CDDP could be important options for the treatment of OC. It is known that Brazil has the greatest biodiversity in the world, with many species still unknown or little studied. Fruit species have high economic values, both in the fresh fruit trade and use as raw materials for agroindustry . The AmaAmong the main preventive strategies for health is chemoprevention, which consists of the use of natural or synthetic chemical agents to prevent, interrupt, stabilize, or reverse the genesis of cancer ,7. Some These fruits are endemic in some ecosystems in Brazil, but produced predominantly in the Amazon region in forest yards and are then harvested by extractivism. They present an increasing economic importance in tropical regions, especially in the field of eccentric juices and desert markets ,13. The Impressive progress is being made in discovering the role of bioactive compounds in reducing the risk of cancer and the underlying biological mechanisms that account for these effects. Epidemiological studies suggest that cancer risk is related to dietary intake of fruits and vegetables that are rich in carotenoids and phenolic compounds . These bME has been described as a good source of lutein and zeaxanthin . BesidesTAP is also a source of phenolic compounds having tannins as the main component. Its antioxidant capacity may also be related to the presence of compounds such as vitamin C and carotenoids. Large amounts of gallic acid and quercetin have been found in TAP, and its pulp can effectively inhibit oxidation, which is attributed to its yellow flavonoids, carotenoids, and chlorophyll ,16,17.Based on the need to develop more effective treatment strategies to reverse chemoresistance and the survival rates in OC patients and the potential properties of ME and TAP as sources of bioactive compounds, this paper investigates the use of extracts from these Amazonian fruits as auxiliary tools to modulate the cell viability and survival of A2780 and ACRP OC cell lines.Pulps of ME and TAP packaged in sealed and labeled plastic bags (1 kg) were supplied by a company from Pará  and stored at a controlled temperature (−18 °C). The frozen pulp was transported in an ice chest containing dry ice to the Laboratory for Analysis of Functional Foods (LAAF-UNIRIO), Rio de Janeiro (Brazil), where they remained frozen (−18 °C) until the moment of analysis.® LD 300, São Carlos, SP, Brazil) for 24 h. After this process, extracts were frozen at −20 °C until use in the experiments [Approximately 250 g of pulp of ME and TAP was extracted with 80 mL of distilled water and then shaken for 2 h. After the pulp maceration period, the aqueous ME and TAP extracts were filtered through Whatman #1 filter paper. The extracts were then frozen at −80 °C in an ultra-freezer and lyophilized  according to Rodriguez-Amaya (2001) [®, Milford, KS, USA), with 80% MeOH , and 20% methyl t-butyl ether as the mobile phase with a column temperature of 33 °C. β-Cryptoxanthin, lutein, zeinoxanthin, α-carotene, β-carotene, and zeaxanthin were quantified.The composition and level of carotenoids in extracts of powdered ME and TAP pulps were determined using high-performance liquid chromatography  . Saponif® 340, Haverhill, MA, USA) in triplicate and the results were expressed as mg gallic acid equivalent (GAE)/mL extract.The Folin–Ciocalteu assay was performed to determine the concentration of total phenolics in the ME and TAP aqueous extract powders. The method was performed as described by Singleton and Rossi (1999) . The extThe antioxidant capacity of the ME and TAP extracts was measured by the ORAC assay, according to Prior and Hoang (2000) . PBS  , by the A reaction mixture of 2.5 mL DPPH methanolic solution (0.06 mM) with 0.5 mL of ME or TAP extracts was mixed completely and left in the dark at a controlled temperature for 60 min. The absorbance of the blend was measured with a spectrophotometer (Turner 340) at 515 nm in triplicate .TEAC assay was performed following the procedure proposed by Rufino et al. (2007) . The ABT2 atmosphere. Stock cultures in flasks were grown to 80% confluence and routinely subcultured. Cell morphology was observed using a Zeiss Observer Z1 microscope and all images were captured using Axio-Vision Rel. 4.8 software . For each experiment, the cells were seeded at 2 × 105 cells/cm2 and 5 × 105 cells/cm2 densities in 96-well plates and 6-well plates.The human ovarian carcinoma cell line (A2780) was obtained from the National Cancer Institute  and the CDDP-resistant cell line (ACRP) was developed using a pulse selection strategy followed by recovery in free media of drugs to mimic the clinical administration of chemotherapy. The two cell lines (A2780 and ACRP) were routinely maintained in an RPMI-1640 medium  supplemented with 10% fetal bovine serum (FBS)  and 1% penicillin, under 5% CO5 in 96-well plates in triplicate and incubated for 24 h following the procedure for cell adhesion. Then the medium was removed and the cells were exposed to nine concentrations of both the extracts (0.01 to 20 mg/mL) for 24 h. The treatment with fruit extracts in combination with CDDP, previously defined at 10 µM and 80 µM, was set for the A2780 and ACRP cell lines, respectively [The cytotoxic effects of ME and TAP extracts were monitored by the 3--2,5-diphenyltetrazolium bromide (MTT) assay. A2780 and ACRP cells were seeded at 2 × 10ectively , while f5 cells/cm2 in a 6-well plate. Cells received treatment with ME and TAP extracts at two concentrations (5 and 20 mg/mL). After incubation for 24 h, the cells were briefly washed with PBS solution and resuspended in 500 μL of Vindelov’s solution containing 0.1% Triton X-100, 0.1% citrate buffer, 0.1 mg/mL RNAse, and 50 mg/mL propionate iodide  [® software .Human ovarian cancer cell lines (A2780 and ACRP) were seeded at 5 × 10MO, USA)  and left® software .The ovarian cancer cell lines were seeded in 6-well plates at concentrations similar to those used for cell cycle analysis. After 24 h of treatment with the extracts at concentrations of 5 and 20 mg/mL, the cells were washed with buffered saline solution (PBS), resuspended in a binding buffer with 5 μL of annexin V FITC and 5 μL of propidium iodide (PI) . Apoptotic and necrotic cells were labeled and quantified in a BD Accuri ™ C6 flow cytometer and analyzed with the CFlown = 9). Data were analyzed using GraphPad Prism . A univariate analysis of variance (ANOVA) and the Tukey’s post-test at a 95% confidence level were used to test cell viability, cell cycle, and apoptosis rates.The results presented are the mean and corresponding standard deviations of three independent experiments performed in triplicate  (1020.39 ± 88.43 μM TE/g), ferric-reducing ability of plasma (FRAP) (1014.71 ± 2.08 μmol ferrous sulfate/g), and Trolox equivalent antioxidant capacity (TEAC) (1620.95 ± 114.65 μmol TE/g), when compared with TAP, which presented mean values of 623.72 ± 38.75 μM TE/g (ORAC), 644.55 ± 10.89 μmol ferrous sulfate/g (FRAP), 78.70 ± 0.28% reduction in  free radicals (DPPH), and 1090.90 ± 296.04 μmol TE/g (TEAC). On the other hand, TAP presented a higher reduction activity in the DPPH assay compared with the ME extract . These vME contained higher levels of total phenolic compounds /mL) compared with TAP (1049.09 ± 95.68 mg GAE/mL), which might explain the antioxidant potential of this extract. Almeida et al. [In other studies, approximately 19 polyphenolic compounds have been identified in murici, including galotannins, quinic acid gallates, proanthocyanidins, quercetin derivatives, and gallium derivatives—substances rarely found in fruits ,28. AuthTAP presented higher levels of total carotenoids (185.92 ± 12.86 μg/g) compared with ME (86.30 ± 8.82 μg/g). Matietto et al.  found toIn the present study, six carotenoids were identified in both the pulps: β-cryptoxanthin, lutein, zeinoxanthin, α- and β-carotene, and zeaxanthin. Among these carotenoids, β-cryptoxanthin, α-carotene, and β-carotene have pro-vitamin A activity . β-cryptCarotenoids are a group of pigments widely distributed in nature that occur in large quantities. These compounds are known for their structural diversity and various biological functions . In fruiTiburshi et al.  found hip < 0.05) (p < 0.05) in A2780 (69.40%) and ACRP (65.54%) cell viability (p > 0.05). When tested at a concentration of 5 mg/mL, TAP promoted a mean reduction of 58.22% in A2780 cell viability. However, it did not promote reduction in A2780 cell viability at the concentrations of 5 and 10 mg/mL compared to ACRP cells, as seen in In order to evaluate the effect of ME and TAP on the viability of the two ovarian cancer cell lines, A2780 and its CDDP-resistant derivative ACRP, these were treated with 0.001–20 mg/mL of each extract for 24 h. ME at a concentration of 20 mg/mL promoted 77.38% and 83.94% decreases in A2780 and ACRP cell viability, respectively, compared with the control group ( < 0.05) . At the iability . MTT tesPessoa et al.  in theirp < 0.05).ME promoted a greater reduction of cell viability in relation to the control than the TAP extract. This decline was also higher in the parental ovarian cancer cell line (A2780) than in the ACRP resistant cell line  and TAP (68.80 ± 5.82%) in A2780 parental cells. The same effect was observed for ME (19.03 ± 3.09%) and TAP (36.60 ± 11.03%) in ACRP, the CDDP-resistant cell line. A decrease of cells in the S and G2/M phases in response to cell treatment with both the extracts was also observed (p < 0.05). The highest concentration of cells in the G0/G1 phase was observed in response to cell treatment with ME in the two cell lines studied (p < 0.05), indicating cell growth arrest of A2780 and ACRP after treatment  and resistant cells (ACRP) demonstrated that the effective dose of CDDP treatment in these situations ranges from 10 µM for A2780 to 80 µM for ACRP cell lines . Based oA2780 cells showed a greater reduction in cell viability after 24 h of treatment with ME and TAP extracts at the concentration of 20 mg/mL. A2780 cells incubated with the lowest concentration of the ME extract (5 mg/mL), as well as those treated with 10 μM of CDDP and a combination of CDDP10 and ME5, presented a similar reduction in cell viability. The treatments combining CDDP10 and TAP extract at the two selected concentrations (5 and 20 mg/mL) showed a similar reduction .Regarding the ACRP cell line, the treatments with the ME and TAP extracts at 20 mg/mL and the treatment combining CDDP80 plus extracts of 20 mg/mL were similar and presented a greater reduction. The treatments with both the extracts at the concentration of 5 mg/mL and combined treatment with CDDP80 and 5 mg/mL ME and TAP extracts were similar to the chemotherapy treatment tested at the concentration of 80 µM. This suggests a preponderant effect of ME and TAP extracts compared to the use of chemotherapy or their concomitant use as a way to promote better results from the synergistic effect of the compounds, especially in cases of resistance to chemotherapy. Similar studies considering fruits from the Amazon region and combined treatment with chemotherapeutic agents in order to inhibit cell viability were not found in the literature.The results indicate that the important bioactive potential of ME and TAP fruits is related to their carotenoid and phenolic content. The levels were higher in the murici extracts, which presented greater action in the cell lines studied, especially in resistance to chemotherapy.From the results obtained, we can conclude that the two Amazonian fruits, ME and TAP, have cytotoxic effects on ovarian cancer cells, leading to cell cycle arrest and apoptosis of diseased cells. The effects of these extracts may open perspectives to design combined approaches to sensitize ovarian cancer cells to current chemotherapeutic drugs such as cisplatin. Therefore, further studies are needed to clarify the putative therapeutic potential of these fruit extracts in ovarian cancer cells.To date, studies reported in the literature about ME and TAP fruit extracts are still scarce, especially of their influence on cancer cell lines. Our study is the first to provide experimental evidence that ME and TAP fruit extracts can inhibit cell viability of parental and CDDP-resistant ovarian cancer cells. The reported studies with these fruit extracts on other cell lines have demonstrated that they are potent antioxidants, providing early evidence that can be used for the development of new chemotherapeutic strategies aiming to prevent the development of several diseases, including cancer.


TRAF1, TNFRSF12A and NFKB2. Our results point to a role of temporal alternative splicing in isoform production, which may alter the outcome of the TNF pathway and impact on tumorigenesis.Alternative splicing plays an important role in numerous cellular processes and aberrant splice decisions are associated with cancer. Although some studies point to a regulation of alternative splicing and its effector mechanisms in a time-dependent manner, the extent and consequences of such a regulation remains poorly understood. In the present work, we investigated the time-dependent production of isoforms in two Hodgkin lymphoma cell lines of different progression stages  compared to a B lymphoblastoid cell line (LCL-HO) with a focus on tumour necrosis factor (TNF) pathway-related elements. For this, we used newly generated time-course RNA-sequencing data from the mentioned cell lines and applied a computational pipeline to identify genes with isoform-switching behaviour in time. We analysed the temporal profiles of the identified events and evaluated in detail the potential functional implications of alterations in isoform expression for the selected top-switching genes. Our data indicate that elements within the TNF pathway undergo a time-dependent variation in isoform production with a putative impact on cell migration, proliferation and apoptosis. These include the genes  Alternative splicing (AS) belongs to one of the key biological processes, which regulates gene expression diversity and function in eukaryotes. In this process, different coding regions of the pre-mRNA are included in the mature mRNA in a combinatorial way. This leads to exon/intron inclusion or exclusion events in the final transcript form of a gene. As a result, AS is able to generate a myriad of functional proteins from the same gene. This affects cellular functions and pathways, and provides the organism an evolutionary advantage to adapt to its changing environment. Remarkably, it was estimated that around 95% of human genes are alternatively spliced ,2. MoreoIn cancer, changes of AS are well studied, and a deregulated AS is reported as one of the hallmarks of cancer ,7. SeverSeveral studies have illustrated a cancer-specific pattern of AS in various cancer types , prostate, colon and lung cancers) ,13,14,15Per1 and CLOCK) [Increasing evidence points to the regulation of several post-transcriptional processes in a time- dependent manner. Genes and proteins hallmarked by circadian (ca. 24 h period) and ultradian (between 8–12 h period) expression were shown to be conserved across several species and regulate several pathways . These id CLOCK) ,19,20. AOne of the pathways for which published data points to a role of AS in the regulation of elements of the signalling cascade is the TNF (tumour necrosis factor) pathway . As demoDespite the increasing relevance of the time-dependent regulation of cellular processes in cancer progression, the impact of circadian regulation in splicing remains poorly understood. Thus, it is of great biological and clinical relevance to identify timely regulated AS events among elements of the TNF pathway in cancer cellular models. Yet, additional studies at the transcriptome level using circadian data sets are still rare and are essential for the investigation of time-dependent AS regulation ,28. In this work, we characterized the extent of a time-dependent isoform generation in elements within the TNF pathway. We generated 33 time-course data sets (sampling every three hours between ZT12 h and ZT42 h) to investigate the time-dependent production of isoforms in two HL cell lines at different stages  compared to a B lymphoblastoid cell line (LCL-HO) with a focus on TNF pathway-related elements. ZT (zeitgeber time) is defined as the time (in hours) after cell synchronization with medium change. Using a computational approach, we identified several genes involved in the TNF pathway to undergo a time-dependent change in isoform expression with potential implications on the output of the pathway. These include TNF receptor-associated factor 1 (TRAF1), TNF receptor superfamily 12A (TNFRSF12A) and the nuclear factor kappa B subunit 2 (NFKB2) with relevant functions in cell proliferation, migration and apoptosis. Our results further emphasize the potential impact of AS in cellular physiology, in a cancer context, and highlight the effect of temporal isoform switching events as mediators of these processes.p-value cut-off of 0.001, and a difference cut-off of 1 as suggested in the TSIS package  [Previous research pointed at the relevance of splicing in cancer, and identified cell-specific AS changes in HL cell lines ,13,29. Idetails) . The difdetails)  for codiWe further investigated the switches between isoforms in time, focusing in the region around the STPs, and tested for potential functional properties of the resulting transcripts at the sequence level . Such prp-value = 8.360 × 10−7) as well as NMD (adjusted (BH) p-value = 1.870 × 10−3) (VEGFA) are involved in a variety of cancers including skin, pancreatic and colon cancer, as well as lung disease according to our enrichment analysis, which is in agreement with previous studies for the mentioned gene [p-value = 7.099 × 10−8). The SLIT/ROBO pathway has important roles in tumorigenesis, cancer progression and metastasis and thus acts as a regulator for multiple oncogenic signalling pathways including the CD20, mTOR , VEGF and EGFR  pathways [p-value = 6.918 × 10−3), interestingly only in the genes with switches that are unique to this cell line  signalling pathway, which is associated with disrupted organogenesis leading to cancer [p-value = 4.542 × 10−2), the SREBP (sterol regulatory element-binding proteins) pathway  and the circadian clock pathway  to be enriched in the L-1236 cell line  . Additio × 10−3) . The genned gene . Howeverpathways . The ROBo cancer . Additioell line . SREBP bell line . For theell line .HNRNPH1 (heterogeneous nuclear ribonucleoprotein H1) shows an absolute difference of expression of 701.44 TPM between the isoforms ENST00000262584.7 and ENST00000526668.5. JCHAIN (joining chain of multimeric IgA and IgM) shows an absolute difference in expression of 107.69 TPM between the isoforms ENST00000510437.5 and ENST00000543780.5 and NME2 (NME/NM23 nucleoside diphosphate kinase 2) shows an absolute difference in expression of 408.09 TPM between the isoforms ENST00000513177.5 and ENST00000512737.5. The HNRNPH1 gene is involved in splicing. It is overexpressed in many cancer types  and is required for cancer growth and development [HNRNPH1 is different over time for the three investigated cell lines  [MYC gene and its isoforms, which are either differentially or ubiquitously expressed [We computed the difference in expression between the switching isoforms with the TSIS package  and geneelopment ,36,37. Nll lines . There i cancer) . NME2 isxpressed . CD44 gene shows an absolute difference in expression between the isoforms ENST00000263398.10 and ENST00000352818.8 of 164.95 TPM. The TNFRSF12A gene shows an absolute difference in expression of 211.08 TPM for the isoforms ENST00000326577.8 and ENST00000341627.5 and the HNRNPA1 (heterogeneous nuclear ribonucleoprotein A1) gene (involved in splicing and mRNA transport) shows an absolute difference in expression of 171 TPM for the isoforms ENST00000340913.10 and ENST00000547566.5. CD44 encodes for a cell-surface glycoprotein and can act both as a growth- and invasiveness-promoting molecule, and as a tumour-suppressing cofactor via its different splice variants [For the HD-MY-Z B cell liNFKB2 gene shows an absolute difference of 84.39 TPM between the isoforms ENST00000189444.10 and ENST00000467116.5, the PPP4C  gene shows an absolute difference of 147.32 TPM between the isoforms ENST00000279387.11 and ENST00000561610.1. The HSPD1 (heat shock protein family D (Hsp60) member 1) gene shows an absolute difference in expression of 348.472 TPM between the isoforms ENST00000388968.7 and ENST00000345042.6. PPP4C mediates the repair process of damaged DNA upon double strand break and HSPD1 is involved in B and T cell activation, among other processes. NFKB2 is a member of NF-KB family of proteins and of the TNF signalling pathway. It is involved in transcription, differentiation, apoptosis and inflammatory response. Altogether, these results indicate that a time-dependent change in isoform expression is present in genes with an impact on vital cellular processes. For the L-1236 cell line C, the NFTNFRSF12A and NFKB2 are related to the TNF pathway. Therefore, we investigated the TNF pathway in detail and detected multiple switches in isoform expression over time in several genes. These included TRAF1 and RACK1 (receptor for activated C kinase 1) in the LCL-HO cell line (RACK1 and SHARPIN (SHANK associated RH domain interactor) in the HD-MY-Z cell line (UBB (ubiquitin B) and RPS27A  for the L-1236 cell line  domains are absent. These are critical for folding and stability and are involved in protein-protein interactions. As mentioned above, the cumulative expression of the gene shows little variations (6.93% from 411.96 TPM to 382.52 TPM) whereas the expression of the isoforms shows strong differences within six hours. These findings point to the potential implication of temporal switching events in isoform expression and its impact in the subsequent protein functions.The presence of switches in isoform expression over time and their concentration at specific time points has potential functional implications on the function of the generated protein and the subsequent alterations in the phenotype of the cell. To investigate this aspect in more detail, we performed an analysis at the sequence level with the R package IsoformSwitchAnalyzeR that allows for the integration of data from tools such as CPAT   for codiell line A. For TNthin 9 h B.The ovethe gene . For theThe abundance of time-dependent changes of isoform expression in the TNF pathway  points tTRAF1 in the LCL-HO cell line, TNFRSF12A and RACK1 in the HD-MY-Z cell line and NFKB2 in the L-1236 cell line, which show significant isoform switching behaviour and can regulate the resulting output of the TNF signalling pathway in this cellular model system.Results from our computational analysis revealed that in all of the investigated cell lines, we found a TNF or TNF-related element with temporal changes in isoform production. We identified In the present study, we characterized the existence of time-dependent AS events in HL cell lines  and a non-tumour B lymphoblastoid cell line (LCL-HO) using a computational approach. Stage-specific patterns of AS and splicing factor expression for HL cells from different tumour stages has previously been reported . The autPublished data points to the regulation of AS, and various post-translational processes by the circadian clock pointing to a time-dependency of such molecular mechanisms with implications in cellular function ,27. To iJCHAIN, NME2 and HNRNPH1 genes (for the LCL-HO cell line), CD44, TNFRSF12A and HNRNPA1 (for the HD-MY-Z cell line) and NFKB2, PPP4C and HSPD1 (for the L-1236 cell line), which have an impact in tumorigenesis. Transcript variants of HNRNPH1 were shown to be differentially involved in tumorigenesis. Overall, positive expression of HNRNPH1 is associated with poor tumour differentiation degree [NME2 gene can act as a metastasis suppressor by altering telomere function and telomere length in lung cancer and fibroblasts [CD44 gene [PPP4C gene is required for the correct DNA damage repair functioning upon a double strand break. Moreover, it was shown to be involved in the TNF and NF-kB pathways. It acts as a signalling component connecting the TNF signal to the JNK pathway [HSPD1 encodes a heat-shock family protein chaperone that exhibits elevated expression in Hodgkin’s and large cell lymphoma and is involved in the B and T cell immune response [We further analysed a reduced set of genes that present splicing switches in a time-dependent manner. In particular, out of the top 20 genes with largest expression difference between the isoforms we identified the n degree ,36. The roblasts ,43. CD44D44 gene . ParticuD44 gene . Cancer D44 gene ,46,47. T pathway . HSPD1 eresponse . Altogether, these genes encode for functionally different isoforms involved in vital cellular and tumorigenic processes and hence, the temporal change in their isoform balance could imply subsequent changes in tumour progression. TNFRSF12A and SHARPIN in the HD-MY-Z cell line, NFKB2 in the L-1236 cell line and TRAF1 in the LCL-HO cell line. The TNF receptor superfamily 12A gene (TNFRSF12A) displays a time-dependent isoform generation in the HD-MY-Z cell line. Surprisingly, one of the transcripts for which a temporal isoform generation was detected undergoes NMD, which may influence its functional outcome in time. In cancer, TNFRSF12A can promote angiogenesis and endothelial cell proliferation. It was reported that TNFRSF12A knockout inhibits hepatocellular carcinoma cell proliferation and migration in vitro [TRAF1) showed a temporal pattern of isoform expression. Interestingly, yeast two-hybrid interaction assays revealed that TNFRSF12A binds to members of the TRAF family including TRAF1 and thereby activates the NF-κB signalling pathway [It was recently reported, that splicing isoforms of the main elements within the TNF pathway can have an impact on both inflammation and cancer and, hence, affect the outcome of the pathway . Among tin vitro ,51. TRAFin vitro ,53. Anotin vitro . Anotherin vitro ,56. In t pathway . Compone pathway . Taken together, the results of our study further reinforce the hypothesis, that AS could potentially fine-tune the outcome of the TNF signalling pathway in a time-dependent manner. This adds a hidden layer of temporal regulation in the TNF pathway that will require further experimental studies to fully validate and further evaluate the impact of our findings in tumorigenesis. 2 atmosphere.The Hodgkin lymphoma cell lines, HD-MY-Z (DSMZ—#ACC 346) and L-1236 (DSMZ—#ACC 530), and the B lymphoblastoid cell line, LCL-HO (DSMZ—#ACC 185) were maintained in RPMI 1640 (Gibco) supplemented with 1% penicillin-streptomycin (Gibco) and 10% foetal bovine serum (Gibco) and incubated at 37 °C with 5% CO5 cells/dish and synchronized by fresh medium addition. Cells were collected every three hours, in triplicates and the pellets were stored at −80 °C. Total RNA was isolated using the RNeasy extraction kit (Qiagen), including DNase digestion, according to manufacturer’s instructions. Cells were lysed with 350 µL RLT buffer and the lysate was homogenized. Total RNA concentration was determined using NanoDrop® ND-1000 ultraviolet–visible (UV–Vis) spectrophotometer and stored at −80 °C. A total of 33 time-course data sets (sampling every three hours between ZT12h and ZT42h) was produced for subsequent sequencing.Cells were seeded in 35 mm dishes with a density of 5 × 10For RNA-sequencing the total RNA was processed by Illumina standard protocols to prepare the mRNA-Seq library, followed by deep sequencing in a bi-directional, 75-base mode on an Illumina NextSeq sequencer. The RNA-sequencing was performed in Genomics Core Facility at the European Molecular Biology Laboratory (EMBL) in Heidelberg.p-value cut-off of 0.05 and Bonferroni Hochberg correction for multiple testing against default “universe” background set of genes with a q-value cut-off of 0.2. Disease ontology enrichment was performed with the disease ontology as ontology of choice and p-value cut-off of 0.05 as well as q-value cut-off of 0.5. In all cases, the top 10 enriched terms (if present) were plotted.Enrichment analysis was performed with the DOSE   without a reduction to the most abundant isoforms. For the analysis of coding functionality, we used a coding potential cut-off of 0.725. For the visualization of pairwise comparisons between the STPs we used a difference cut-off of 0.1. Genes with single isoform are removed from the pairwise STP analysis. For the detection of splicing switches over time the TSIS R package (ver. 0.2.0) was used with “mean” as the detection method and a spline degree of 4 to focus on isoforms with a period close to 24 h. Isoform switches were visualized with the TSIS  package d is the average difference of abundances between iiso and jiso in the interval kI, k = 1,2 is defined as: kI| is the number of samples in interval kI and exp is the expression of iiso of sample mIks in interval kI. Computation of abundance differences : MetricThe RNA-sequencing data was submitted to the GEO repository and will be released upon publication of the manuscript.


Overview of current understanding of epigenetic alterations after abiotic stresses during reproductive development in cereals.Abiotic stresses, including heat, drought, cold, flooding, and salinity, negatively impact crop productivity. Various stages during reproductive development are especially sensitive to environmental stresses, which may lead to complete sterility and severe yield losses. Plants exhibit diverse responses to ameliorate stress damage. Changes in DNA methylation, histone modification as well as regulation of small RNA and long noncoding RNA pathways have been shown to represent key modulators in plant stress responses. During reproductive development in cereals, various protein complexes controlling histone and DNA methylation have been identified, revealing conserved and novel mechanisms regulating abiotic stress responses in cereals and other plant species. New findings highlight the role of transposable elements during stress periods. Here, we review our current understanding of epigenetic stress responses during male and female gametophyte formation (germline development), fertilization, early seed devolvement, and seed maturation in cereals. An integrative model of epigenetic responses during reproductive development in cereals is proposed, emphasizing the role of DNA methylation and histone modifications during abiotic stresses. With the human population continuously growing, the demand for food supply is increasing exponentially. Unfortunately, production of cereal grains, including some of the most important crops such as maize, wheat, rice, barley, rye, and oats, is highly vulnerable to abiotic stresses - and long noncoding RNA (lncRNA)-associated regulatory pathways are affected during plant development under stress conditions. These include stages of reproductive development such as gametogenesis and seed development  epigenetic alterations during pre-fertilization processes . Then, we discuss (2) fertilization mechanisms, gamete activation, and early seed development involving pro-embryo and syncytium formation as well as endosperm cellularization. Finally, we cover a stage known as (3) seed maturation, in which starch accumulation, desiccation, and seed dormancy are major processes taking place. Even though reproductive development can be subdivided into several other stages, recent reports have shown the importance of the aforementioned stages or phases as determinant for productivity in cereals , which promotes male germline cell fate and meristem growth  were lacking, suggesting that stress-induced mis-regulation of MSCA1 causes the absence of phasiRNAs in anthers and thus affects a number of unknown target genes . When MAC1 is expressed, a small protein is generated that induces adjacent cells to differentiate into supportive tissues  showed gradual degeneration of epidermal and archesporial cells resulting in anther abortion as a consequence of transcriptional inhibition , which encodes a germline-specific AGO protein that preferentially binds 21-nt phasiRNAs derived generally from intergenic regions. Additionally, MEL1 plays a central role in the progression of meiosis beyond the leptotene stage  from pre-meiosis to meiosis stage is controlled by an increase in di-methylation of lysine 9 of histone 3 (H3K9me2) and a decrease in acetylation of lysine 9 of histone 3 (H3K9) as well as phosphorylation of histone 3 (H3S10) (Liu and Nonomura ZmAGO18b) was pointed out as a possible candidate based on its transcriptional profile . A detailed analysis of QTLs revealed the presence of miRNA targets associated with ABA-responsive genes. Additionally, suppressor of the G2 allele of skp1 (SGT1), a direct target of miRNA166e, was also mapped within the qHT-8 locus  concentration in pollen grains correlates well with pollen sterility and decreased yield  where they germinate and grow toward the ovule to deliver its sperm cell cargo. To which extent this so-called progamic phase of reproduction is affected by abiotic stresses is almost completely unclear. A comparison between pre- and post-pollinated silks in maize identified 30 conserved miRNA families  expression was regulated in a biphasic manner. First, levels of histone H3K4 changed from a di- to a tri-methylation state followed by an increase in acetylation of H3  represents a major regulator of early seed development. During endosperm development in cereals, several active members of PRC2 have been identified  insertion into the 3′-UTR of HSP16.9 affecting its transcription rate  required for maternal allele demethylation and imprinting in the endosperm was reported to be involved in responses to stress. In a drought-tolerant barley variety, increased A recent study performed during rice seed development detected a strong increase in acetylation levels of proteins during seed differentiation and maturation  gene, which is a target of the RdDM pathway (Surdonja et al. Additionally, RdDM pathways have been shown to regulate many developmental processes including seed maturation in cereals (Lanciano and Mirouze Epigenetic mechanisms controlling reproductive development have been shown in the past decade as pivotal regulators of stress responses in cereals. Development and function of female and especially male gametophytes appear to be especially susceptible to abiotic stresses. DNA methylation and small regulatory RNAs including phasiRNAs, lncRNAs, and miRNAs have been shown as repressors of germline initiator genes. Mis-regulation of germline-specific genes during abiotic stresses seems to be a consequence of the lack of the aforementioned epigenetic regulators Fig. .It is still unclear whether the fertilization process itself is affected by stresses and if so, which epigenetic components are the main targets. However, since histone variants are exchanged during zygotic gene activation at non-stress conditions and DNA methylation of imprinted genes is active immediately after fertilization, we speculate that this stage is also affected by abiotic stresses. Proper activity of the PRC2 complex is very critical for seed formation during early stages involved in cell/nuclei divisions, endosperm cellularization, and embryo patterning Fig. . AbioticThese above-described findings now provide exciting prospects since a large number of these processes are crucial for maintenance of yield and reproductive success under abiotic stress conditions. In conclusion, even though plants are exposed to abiotic stresses throughout their life cycle, reproductive development is more vulnerable to abiotic stresses compared to vegetative developmental stages as stress may terminate development and lead to complete sterility. The systematic study of epigenetic regulatory mechanisms under abiotic stress conditions during reproductive development needs to be extended and approached under field conditions, since multiple stresses are frequently coexistent. Inheritable epigenetic changes such as small regulatory mechanisms, histone variants, and DNA methylation are also unexplored in cereals in a trans-generational memory context, since these epigenetic variations might improve stress resilience in the offspring.Finally, although new tissue- and cell-specific methodologies including fluorescence-activated cell sorting, ATAC-seq, and methylome sequencing (e.g., Begcy and Dresselhaus


Leymus chinensis, a perennial rhizomatous grass, and conducted a series of field experiments in situ, including grading sampling of reproductive ramets and different connection forms of vegetative ramets labeled with 15N at four reproductive stages. Reproductive ramet biomass, inflorescence biomass, seed number, seed-setting percentage, reproductive allocation, and reallocation significantly increased with an increase in the number of vegetative ramets connected to tillering nodes, and the plasticity indexes of these six phenotypic characteristics showed similar increasing trends. The amount of nutrients supplied from the connected vegetative ramets to the reproductive ramets was significantly affected by the transfer direction, reproductive stage, and position order of the vegetative ramets. Throughout the sexual reproduction stage, nutrients were preferentially transferred to the acropetal reproductive ramet in L. chinensis populations. The amount of nutrients supplied from the connected vegetative ramets to the reproductive ramets at the milk-ripe stage, when sexual reproduction was most vigorous, was significantly larger than that at other reproductive stages. The amount of nutrients supplied from the spacer vegetative ramet to the acropetal reproductive ramet was significantly larger than that to the basipetal reproductive ramet. The closer the vegetative ramet was to the reproductive ramet, the more nutrients were supplied; the amount of nutrients supplied was significantly negatively related to the position order of the vegetative ramet. We identified the determinant of plasticity in sexual reproduction in clonal plants in a homogeneous environment: physiological integration between ramets within clones. Our results are vital for better understanding the adaptation of populations and even the evolution of species of clonal plants.Phenotypic plasticity is considered a major mechanism that allows plants to adapt to heterogeneous environments. The physiological integration between the interconnected rhizomes or stolons of clonal plants influences the plasticity of such plants in heterogeneous environments. However, the determinants of plasticity of reproductive ramets in clonal plants in homogeneous environments are unclear. Here, we chose  Phenotypic plasticity, or the ability of a given genotype to generate various phenotypes under different environmental conditions , is consClonal plants are widely distributed in numerous ecosystems, such as grasslands and wetlands , and thePrevious studies concerning the effect of physiological integration on the plasticity of clonal plants were mostly carried out in heterogeneous environments. Moreover, what has been confirmed relies on the heterogeneity of the environment . CurrentLeymus chinensis, a typical clonal plant with vigorous belowground rhizomes  to verify that the plasticity in sexual reproduction in L. chinensis clones can be induced by differences in nutrient supply from the connected vegetative ramets in a natural homogeneous environment and b) to reveal the patterns of nutrient supply from the connected vegetative ramets to reproductive ramets in L. chinensis clones. We hypothesize that 1) the phenotypic characteristics and plasticity in these characteristics of reproductive ramets will increase with an increase in the number of vegetative ramets connected to tillering nodes; 2) the amount of nutrients supplied from vegetative ramets with various connection forms to the reproductive ramets will be largest at the vigorous milk-ripe stage; 3) the amount of nutrients supplied from spacer vegetative ramets to acropetal reproductive ramets will be much larger than that to basipetal reproductive ramets; and 4) the closer the vegetative ramet is to the reproductive ramet, the more nutrients will be translocated.In this study, we chose The study was conducted at the Grassland Ecological Research Station of Northeast Normal University in the southern Songnen Plain in Changling County, Jilin Province, Northeast China , in 2018, the fourth year after the experimental plots were set up. This area is characterized by a semi-humid, semi-arid, and temperate continental monsoonal climate with hot and rainy summers and cold and dry winters. The annual mean temperature varies from 4.6 to 6.4°C. The number of days with an average temperature ≥10°C ranges from 120 to 140. The annual accumulated temperature varies from 3,000 to 3,500°C. The annual mean precipitation ranges from 300 to 450 mm, with the majority concentrated in June-September, and the annual evaporation varies from 1,200 to 1,400 mm. The frost-free period is approximately 130–165 days .L. chinensis (Trin.) Tzvel., a perennial rhizomatous C3 grass, is widely distributed in the eastern Eurasian steppe, including northwestern Siberia, western North Korea, the People’s Republic of Mongolia, the Inner Mongolian Plateau, and the Northeast Plain of China . Most often, only one of the ramets interconnected by tillering nodes undergoes sexual reproduction, flowering and seed set, called reproductive ramets; the others only conduct vegetative growth and do not produce flowers, called vegetative ramets. In the Songnen grassland, L. chinensis usually begins turning green in early April, heads in the middle or later period of May, flowers in June, and ripens seeds in mid-July , and adjacent plots were at least 2 m apart. Similarly sized vegetative ramets of L. chinensis  were collected from a natural clone in the study area, and a group of nine ramets was transplanted into each plot with rows 0.5 m apart and ramets 0.5 m apart. All the experimental plots were manually irrigated for several days after transplanting to ensure that the ramets survived. No fertilizer was added to the soil. L. chinensis ramets were not obviously affected by diseases or insect pests throughout the study, and all other plants were regularly pulled by hand from 2015 to 2018. The soil type was sandy loam. The soil of the top 20-cm-thick layer was homogenous, with a total N of 1.01 g kg−1, an organic C of 6.23 g kg−1, a total P of 0.74 g kg−1, a pH of 8.37, and an electrical conductivity of 70.85 μS cm−1.In May of 2015, a total of 30 experimental plots were established. Each plot had an area of 4 mL. chinensis in 2018. Reproductive ramets whose inflorescence top reached approximately 2 cm over the flag leaf sheath and that were interconnected with 1, 2, 3, and ≥4 vegetative ramets by tillering nodes were marked with tags. For each number in the gradient, in each plot, only one reproductive ramet was marked, for a total of 25 reproductive ramets in 25 plots. Five plots were disqualified because the desired number gradient was unavailable. All marked reproductive ramets were harvested together at the dough stage. Reproductive ramet height, floret number, and seed number were measured. Each reproductive ramet was separated into leaves, stem, inflorescence, and seeds. Dry biomass, including all parts of a reproductive ramet, was determined after oven-drying to a constant weight at 65°C.To investigate the effects of the number of vegetative ramets connected to tillering nodes on the phenotypic characteristics of reproductive ramets in a natural homogeneous environment, a marking manipulation was conducted at the early heading stage of in situ-labeling experiment , reproductive stage  was applied, with a urea concentration of 2% g ml−1 and a 15N abundance of 5.18%. The urea solution was daubed gently with a small paintbrush from the base to the tip of the leaf surface to ensure that the total solution was smeared evenly on both sides of the leaf. During brushing, leaves were held with forceps. Labeling was applied once per day over three consecutive days, and meticulous operation with extra care was required to prevent the urea solution from contacting any part of the reproductive ramet or soil. For the control treatment, the 15N-labeled urea solution was replaced by an equal volume of distilled water.Each δ) notion and expressed as follows:At each reproductive stage, the reproductive ramets that were connected to the labeled vegetative ramets were harvested 2 days after labeling. Each ramet was separated into leaf blades, sheath, stem, and inflorescence. The enzyme activity of each part was halted at 105°C for 30 min, and the tissue was dried to a constant weight at 65°C and ground to a fine powder using a ball mill . Approximately 3 mg of sample powder was loaded into a capsule and analyzed using an Elementar Vario EL Cube  coupled to an isotope ratio mass spectrometer , with an overall precision greater than 0.2‰. The isotope value was used the standard delta .where R15N from labeling, the F-ratio was calculated as follows:To calculate the amount of 15N in each sample was then calculated as follows:The amount of translocated Wdry is the dry weight of a sample, B is the nitrogen content per unit mass of a sample, FS is the F-ratio of a sample, and FU is the mean F-ratio of four unlabeled control ramets for each harvest.where P < 0.05.All statistical analyses were performed using SPSS 22.0 software . All data were tested for a normal distribution and homogeneous variances through a Kolmogorov-Smirnov test and Levene’s test, respectively. The significance level was set at One-way ANOVA was conducted to assess the effect of the number of connected vegetative ramets on the phenotypic characteristics of reproductive ramets. The seed-setting percentage was calculated as the percentage of seeds relative to the number of florets, reproductive allocation was calculated as the percentage of inflorescence biomass relative to ramet biomass, and reproductive reallocation was calculated as the percentage of seed biomass relative to ramet biomass. To evaluate the phenotypic plasticity in each characteristic of reproductive ramets interconnected with different number grades of vegetative ramets by tillering nodes, the phenotypic plasticity index (PPI) was calculated as  follows:T is the characteristic mean value in each number gradient.where 15N in the leaf blade, sheath, stem, inflorescence, and whole ramet of the reproductive ramet and to investigate the effects of position order and reproductive stage on the amount of translocated 15N in the leaf blade, sheath, stem, inflorescence, and whole ramet of the reproductive ramet. All data on the amount of translocated 15N were log-transformed to meet model assumptions. An independent samples t-test and Duncan’s multiple-range test were used to test for significant differences between means of two and more than two groups, respectively. Estimation of regression curves was used to quantify the relationships between the amount of translocated 15N in the leaf blade, sheath, stem, inflorescence, and whole ramet of the reproductive ramet and position order of the acropetal vegetative ramet at the heading stage, flowering stage, milk-ripe stage and full-ripe stage with linear, exponential, power, and logarithmic functions. The model with the lowest Akaike information criterion (AIC) value was selected as the best  or five members (one reproductive ramet and four vegetative ramets connected via one and the same rhizome). In the present study, we detected significant effects of transfer direction on the amount of translocated 15N in the leaf blade, sheath, stem, inflorescence, and whole ramet of the reproductive ramet in L. chinensis with a three-member clonal system  and basipetal translocation  . Most st a clone . Previoue ramets . Howeverl system Table 3.via one and the same rhizome, significant effects of the position order of the connected vegetative ramet on the amount of translocated 15N were found in the leaf blade, sheath, stem, inflorescence, and whole ramet of basipetal reproductive ramets of L. chinensis , National Natural Science Foundation of China (31672471 and 31670427), and the Program for Introducing Talents to Universities (B16011).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Casearia sylvestris , popularly known as “guaçatonga”, is a plant widely used in folk medicine to treat various diseases, including cancer. The present work deals with the chemical composition as well as the cytotoxic evaluation of its essential oil, its main constituent and derivatives. Thus, the crude essential oil from leaves of C. sylvestris was obtained using a Clevenger type apparatus and analyzed by GC/MS. This analysis afforded the identification of 23 substances, 13 of which corresponded to 98.73% of the total oil composition, with sesquiterpene α-zingiberene accounting for 50% of the oil. The essential oil was evaluated for cytotoxic activity against several tumor cell lines, giving IC50 values ranging from 12 to 153 μg/mL. Pure α-zingiberene, isolated from essential oil, was also evaluated against the tumor cell lines showing activity for HeLa, U-87, Siha and HL60 cell lines, but with IC50 values higher than those determined for the crude essential oil. Aiming to evaluate the effect of the double bonds of α-zingiberene on the cytotoxic activity, partially hydrogenated α-zingiberene (PHZ) and fully hydrogenated α-zingiberene (THZ) derivatives were obtained. For the partially hydrogenated derivative only cytotoxic activity to the B16F10-Nex2 cell line (IC50 65μg/mL) was detected, while totally hydrogenated derivative showed cytotoxic activity for almost all cell lines, with B16F10-Nex2 and MCF-7 as exceptions and with IC50 values ranging from 34 to 65 μg/mL. These results indicate that cytotoxic activity is related with the state of oxidation of compound. Casearia was traditionally regarded as belonging to the family Flacourtiaceae. However phylogenetic and chemical studies, besides ecological and morphological observations showed that Flacoutiaceae is, in fact, composed by two clades, with the genus Casearia belonging to the Salicaceae + cation peak at m/z 123. A further fragmentation of this peak with loss of ethylene produced the base peak of spectrum m/z 95 , suggesting that this sesquiterpene may be primarily responsible for the cytotoxic activity against this particular cell line.The crude oil displayed activity against all cell lines tested with IC50 of 65.2 μg/mL, and IC50 values greater than 200 μg/mL for the other tumor cell lines, indicating that PHZ has low cytotoxic activity against these tumor cells.Additionally, it was observed that at the tested concentrations PHZ was only active against the B16F10-Nex2 cell line, with an IC50 values ranging from 34–65 μg/mL. The higher lipophilicity of totally hydrogenated zingiberene, compared to other compounds, could be responsible for enhanced cell penetration through the plasma membrane. The totally hydrogenated zingiberene (THZ) showed cytotoxic activity against almost cell lines, except for B16F10-Nex2 and MCF-7, with ICC. sylvestris, Salvador et al. [C. lasiophylla were cytotoxic against tumor cell lines U251, UACC-62, MCF-7, NC1-ADR/RES, NCI-H460, PC03, OVCAR-3, HT-29 K562. Silva et al. [C. sylvestris collected in Campinas against several tumor cell lines . However, these oils were shown to be composed by bicyclogermacrene and β-caryophyllene as main compounds, a different profile of that detected in the present work.Although there are no studies concernng the cytotoxic activity of sesquiterpenes isolated from oil of r et al.  showed ta et al. , also shCasearia sylvestris with no deformation and degradation by exogenous factors were selected and collected with the aid of sterile pliers from a single tree at the Atlantic Forest area in São Paulo city, SP, Brazil , in October, 2012.Leaves of C. sylvestris were extracted over 5 h by steam distillation in a Clevenger type apparatus to afford 2.9 g of crude essential oil. The oil was analyzed by GC-FID and GC-LR-EI-MS using a RtX-5 capillary column. The identification of the individual compounds was performed by comparison of retention indexes  and comparison of recorded mass spectra with those available in the system library [2Cl2 in a split mode (1:10) employing helium as the carrier gas (1 mL/min) under the following conditions: injector and detector temperatures of 220 °C and 250 °C, respectively; oven programmed temperature from 40–240 °C at 3 °C/min, holding 5 min at 240 °C. GC-FID was performed in quantitative analysis. The percentage compositions of the oil samples were computed by internal normalization from the GC peak areas without using correction for response factors. GC-LR-EI-MS analysis was conducted on a Shimadzu GC-17A chromatograph interfaced with a MS-QP-5050A mass spectrometer. Helium was used as the carrier gas. The LR-EI-MS operating conditions were an ionization voltage of 70 eV and an ion source temperature of 230 °C with the same conditions described above.Fresh leaves  of  library . GC chro2 gel soaked with AgNO3 (10%) column (63 cm × 5 cm i.d.) chromatography eluted with CH2Cl2-acetone in proportions of 99:1, 95:5 and 90:10 (200 mL for each eluent) to afford 30 fractions which were individually analyzed using GC-FID and then pooled into nine groups (A to I). Fraction F was composed of pure α-zingiberene (203 mg).Part of crude oil (2.0 g) was subjected to flash chromatography on SiOα-Zingiberene. 1H-NMR : 2.01 m , 5.38 brs , 5.57 dd , 6.70 dd , 2.14 m , 1.16 m , 1.48 m , 1.99 m , 5.02 brt , 1.61 s , 1.64 s , 0.83 d , 1.68 d . 13C-NMR : 26.2 (C-1), 120.6 (C-2), 131.6 (C-3), 128.1 (C-4), 131.3 (C-5), 38.3 (C-6), 34.5 (C-7), 36.3 (C-8), 24.7 (C-9), 125.0 (C-10), 131.4 (C-11), 26.2 (C-12), 17.9 (C-13), 17.8 (C-14), 21.3 (C-15). LR-EI-MS (70 eV): m/z (rel. int.): 204 (9), 145 (26), 132 (90), 119 (98), 105 (62), 91 (43), 55 (47).2/g) and hexane (5 mL) were added. After addition of H2 (8 atm), the mixture were stirred for 3 h at 90 °C. Then, the reactor was cooled and the product was filtered [1H-NMR : 1.60 m , 2.01 m , 5.32 m , 2.04 m , 1.63 m , 1.16 m , 1.25 m , 1.23 m , 1.62 m , 0.91 d , 0.86 d , 1.66 s . This data suggests that the remaining double bond is located as shown in 2, Ni-Raney 20%, 30 mg, 120 °C, 3 h) to obtain 60 mg (65%) of the fully saturated product THZ. In a high-pressure reactor (stainless steel), pure α-zingiberene , Ni-Raney catalyst . The melanotic B16F10-Nex2 subline, characterized at the Experimental Oncology Unit , is characterized by low immunogenicity and moderate virulence. Human melanoma (A2058), glioblastome (U87), leukemia (HL-60), uterus carcinoma (Siha) and breast cancer (MCF-7) cell lines were obtained from the Ludwig Institute for Cancer Research. Human cervical carcinoma (HeLa) was acquired from Dr. Hugo Pequeno Monteiro (UNIFESP).C. sylvestris, as well as pure α-zingiberene, zingiberene partially hydrogenated and zingiberane, were resuspended in dimethyl sulfoxide (DMSO) at a final concentration of 10 mg/mL, diluted in RPMI medium containing 10% fetal calf serum ranging from 100 to 0 µg/mL and incubated with 1 × 104 cells in a 96-well plate. After 18 h of incubation, cell viability was measured using the Cell Proliferation Kit I (MTT) , an MTT-based colorimetric assay [The essential oils extracted from leaves of ic assay . ReadingC. sylvestris analyzed in this work showed a different profile in comparison to other previously analyzed oils, being composed by approximately 50% of α-zingiberene. The cytotoxic evaluation displayed that the crude essential oil was active against all cell lines tested, while purified α-zingiberene showed less activity, indicating that there may be synergism of zingiberene with other substances present in the crude oil for activity. For PHZ, the cytotoxic activity decreased, while THZ showed cytotoxic activity against almost all the tested cell lines. These findings suggests that oil from C. sylvestris may be an interesting agent for cancer treatment; however additional studies must be done including isolation of active compound, mechanism of action on tumor cells as well as experimental assays.Essential oil from leaves of


Neurofibromatosis 1 and 2, although involving two different tumour suppressor genes , are both cancer predisposition syndromes that disproportionately affect cells of neural crest origin. New therapeutic approaches for both NF1 and NF2 are badly needed. In promising previous work we demonstrated that two non-steroidal analogues of 2-methoxy-oestradiol (2ME2), STX3451-7-methoxy-6-sulfamoyloxy-1,2,3,4-tetrahydroisoquinoline), and STX2895 -1,2,3,4-tetrahydroisoquinoline) reduced tumour cell growth and induced apoptosis in malignant and benign human Neurofibromatosis 1 (NF1) tumour cells. In earlier NF1 mechanism of action studies we found that in addition to their effects on non-classical hormone-sensitive pathways, STX agents acted on the actin- and myosin-cytoskeleton, as well as PI3Kinase and MTOR signaling pathways. Tumour growth in NF2 cells is affected by different inhibitors from those affecting NF1 growth pathways: specifically, NF2 cells are affected by merlin-downstream pathway inhibitors. Because Merlin, the affected tumour suppressor gene in NF2, is also known to be involved in stabilizing membrane-cytoskeletal complexes, as well as in cell proliferation, and apoptosis, we looked for potentially common mechanisms of action in the agents’ effects on NF1 and NF2. We set out to determine whether STX agents could therefore also provide a prospective avenue for treatment of NF2.STX3451 and STX2895 were tested in dose-dependent studies for their effects on growth parameters of malignant and benign NF2 human tumour cell lines in vitro. The mechanisms of action of STX3451 and STX2895 were also analysed.Although neither of the agents tested affected cell growth or apoptosis in the NF2 tumour cell lines tested through the same mechanisms by which they affect these parameters in NF1 tumour cell lines, both agents disrupted actin- and myosin-based cytoskeletal structures in NF2 cell lines, with subsequent effects on growth and cell death.Both STX3451 and STX2895 provide new approaches for inducing cell death and lowering tumour burden in NF2 as well as in NF1, which both have limited treatment options. Both Neurofibromatosis 1 and 2 (NF1 and NF2) are disorders characterized by the formation of tumours of the peripheral and central nervous system , 4.DCAF1 [Neurofibromatosis type II (NF2) is associated with loss-of-function mutations in the NF2 gene that encodes the multi-functional protein, Merlin (Moesin-Ezrin-Radixin-like protein) , also knDCAF1 .NF2 affects one in 25,000–30,000 live births worldwide. A hallmark of the disease is the formation of bilateral vestibular Schwannomas, as well as the formation of multiple meningiomas, extramedullary spinal tumours, and ependymomas . UncontrCurrent treatment options for NF2 tumours include surgical resection of either part of or the complete tumour, which is difficult to perform without damaging nerves. Stereotactic radiosurgery is also an option, however the risk of malignant transformation rises several years post-surgery , 21. AltPrevious studies from our laboratories  demonstr50 of 40 and 52 nM on DU-145 human prostate and MDA MB-231 breast cancer cell lines, respectively [The two non-steroidal 2ME2 analogues, STX3451 -7-methoxy-6-sulfamoyloxy-1,2,3,4-tetrahydroisoquinoline) and STX2895 -1,2,3,4-tetrahydroisoquinoline) . Recentectively .Fig. 1CHere, we show that NF2 status plays a role in the effectiveness of these non-steroidal 2ME2 derivatives on cell viability in vitro. STX2895 and STX3451 treatment on NF2 null (−/−) cell lines induces nuclear fragmentation, microtubular disruption, inhibition of cellular migration/wound healing, and can induce apoptosis in both benign and malignant NF2 tumour-derived cells.−/− cell line from a 41-year-old woman [+/+ cell line derived from the brain of 61-year-old man [2 incubator [NF2-null malignant meningioma KT21-MG1-Luc5D cells  and theld woman ) were geld woman ) were ki-old man ) was obtSTX3451 and STX2895 were synthesized according to previously published protocols , 45. Pak4/well) determined by a hemocytometer. STX3451 or STX2895 at concentrations of 100 nM, 300 nM, 600 nM, and 1000 nM was dissolved in vehicle  and were added to the wells 2 hours after plating the cells. The final concentration of DMSO was 1%. PF3758309 (PF) and Frax1036 concentrations ranged from 0.03 μM to 20 μM and from 2.5 μM to 25 μM, respectively. The final concentration of DMSO was 2.5%. STX3451, STX2895, PF, or Frax1036 was replenished after 2 days of culture. Cell growth was assessed using CellTiter 96  [Cells were plated in 96-well Primaria™ plates  , 48.40,000–80,000 cells plated on 0.1% porcine gel-coated coverslips were treated with DMSO, STX3451, STX2895, PF3758309, or Frax1036 for various time periods. Immunocytochemistry and counter-staining with DAPI , were carried out as reported previously [5 cells were plated in a 10 mm cell culture dish and allowed to grow to confluence. Wounds were made using a ruler to maintain a straight edge and a 200 μl pipette tip applied to the bottom of the dish to remove the cells. The culture plates were then washed twice with PBS. Fresh medium with DMSO, STX3451, or STX2895 was added to the dishes. STX3451 and STX2895 were used at 300 nM for KT21, Ben-Men-1, and HEI-193 cells, 1000 nM for IOMM-Lee and BJ cells. Photographs of the wounds and subsequent cell migration and “wound filling” were taken using a Nikon SMZ1500 dissecting microscope and digital camera  at the time points indicated. The dimensions of the “wound” were measured with ImageJ software.The ability of cells to migrate was assessed by wound-healing assays , 50. 2 ×Cell lysates were collected after 24 h treatments with DMSO, STX3451, STX2895, PF, or Frax1036. Western blotting was performed as described in the studies on NF1 . MembranThe STX therapeutic agents tested are multifaceted in their mechanisms of action. This study was directed at discovering whether the mechanisms of action that make the STX agents promising potential therapeutics for the treatment of NF1 would also apply to the potential treatment of NF2. Our previous studies concluded that the agents’ effects on the growth of and induction of apoptosis in human NF1 tumour cells were both through NF1-specific pathways and through significant effects on both the actin-based and myosin-based cytoskeleton . The sam−/− benign meningioma cells), CH157MN (NF2−/− meningioma cells), HEI-193 (NF2−/− benign vestibular Schwannoma cells), KT21  and IOMM-Lee  to the individual agents or to a combination of the two. Our results showed that both agents either completely blocked cell proliferation or induced cell death in NF2−/− cells whether the cell lines were derived from benign or malignant tumours  testing whether the STX agents act mechanistically on the same pathways on which we found them to be very effective on growth and apoptosis in human NF1 tumour cell lines, we currently have been able to detect NO significant effects of either of the agents on the growth, morphology of NF2 cell lines through the pathways that are known to be important for the growth of or cell death in NF1 human tumour cell lines . This iOur previous studies on NF1 cell lines demonstrated that STX3451 caused nuclear fragmentation in both the NF1-null malignant tumour cell line ST88 and in benign plexiform neurofibroma (PNF) cells, although this result was less significant for PNF cells .We therefore examined whether this effect was also observed in our studies of NF2-null tumour cells. DAPI staining revealed that treatment with both STX3451 and STX2895 resulted in a dramatic increase in nuclear fragmentation, which was seen in both KT21 and Ben-Men-1 cell lines 48 h after treatment; with STX3451being more effective than STX2895  were apoptotic at 72 h, indicating that these analogues of 2ME2 also have effects on cell growth/apoptosis in the benign Ben-Men-1 cells. However, the effects seen are much less than those seen when either STX agent was used to treat malignant KT21 cells. By contrast, 72 h treatment with PF resulted in 20% annexin V-positive cells at very low concentration, the IC50 for KT21 was 29.25 nM . Frax103Finally, STX3451 and STX2895 at 1000 nM did not significantly increase the percentage of annexin V-positive BJ cells at 72 h, consistent with the results seen in the cell viability assays (discussed above). At concentrations that effectively induced apoptosis in NF2 tumour cells, STX3451 and STX2895 did not cause such programmed cell death in BJ fibroblasts. PF at 5 μM also did not increase the percentage of annexin V-positive BJ cells, whilst Frax1036 at 10 μM induced 55% cells to go through apoptosis.STX3451 has been demonstrated to depolymerize microtubules in both NF1 ST88 and PNF cell lines . Our curOur results from annexin V and α-tubulin staining Fig.  suggest . We found that the cell lines, which we evaluated migrated to fill the wound gaps at different rates, and that STX3451 and STX2895 inhibited wound healing in the NF2 cells with different kinetics. The untreated control Ben-Men-1 cells were able to fill in the gaps by 4 days after the wound was made. However, cells treated with either STX3451 or STX2895 also filled in the gap, but at a much slower pace  [We previously demonstrated that STX3451 induces apoptosis in human NF1-deficient ST88 and S462 malignant peripheral nerve sheath tumour (MPNST) cell lines at very low concentration 300 nM), whilst arresting cell proliferation in an NF10 nM, whiIntriguingly, our results showed that STX3451 and STX2895 were not as effective against a permanent NF2 wild type malignant meningioma cell line, IOMM-Lee, indicating that NF2 status could be important for the effects of these compounds. Our results also showed that these 2ME2 analogs were more potent than Pak inhibitors in inducing cell death. However, their mechanistic effects on the cells were different: STX3451 and STX2895 affect microtubules  and/or meningiomas has not been well-studied. It has been shown that in sporadic VS, both oestrogen receptor (ER) and progesterone receptor (PR) were up-regulated, whilst in NF2-related VS, PR was down-regulated [Our previous study showed that the non-steroidal analog of 2ME2, STX3451, caused microtubule depolymerization and cell death in NF1 deficient tumour cells, even in the presence of elevated hormones . Whetheregulated . Amongstegulated . PR-negaegulated , and expegulated . Howeveregulated , 61 and egulated . Treatmeegulated . TherefoSince Merlin affects several key signalling pathways in the cytoplasm and nucleus, including PI3K signaling, mTORC1, the RAS and Src pathways, the Hippo kinase cascade, and CRL4-DCAF , inhibitSo far, none of the drugs tested in vitro or in pre-clinical or clinical trials has been effective on NF2 meningioma. Although, another mTOR inhibitor, AZD-2014, and a combination of smoothened receptor inhibitor, vismodegib, and FAK inhibitor, vistusertib, are in phase II trials. AR-42, a histone deacetylase inhibitor targeting CRL4-DCAF, which induced meningioma cell apoptosis in vitro and in xenografts, is also in phase I trials. Even if some inhibitors for NF2/Merlin signalling pathways have promising efficacy, drug resistance to these inhibitors may eventually occur and alternative choices are necessary. The microtubule-disrupting non-steroidal 2ME2 analogs could provide another option for treating NF2-related tumours, both vestibular Schwannomas and meningiomas, either alone, or in conjunction with other agents. Pre-clinical studies using several similar THIQ-based molecules have been shown to be safe and efficacious for reducing the size of human breast cancer  and melaBoth STX3451 and STX2895 provide new approaches for inducing cell death and lowering tumour burden in NF2 as well as in NF1, which both have limited treatment options.


Skeletonema marinoi have selective cytotoxic activity against the haematological cancer cell line U-937 and colon cancer cell line HCT-116 compared to normal MePR-2B cells. LC-MS analysis of the raw extract revealed that in their natural form, MAGs occur as 2-monoacyl derivatives and include mainly C16 and C20 analogues, but they are converted into the corresponding 1-isomers during purification processes. Pure compounds along with the synthetic 1-monoarachidonoylglycerol tested on HCT-116 and U-937 tumor cell lines induced cell death via apoptosis. The mechanism of action was investigated, and we show that it involves the induction of apoptosis through caspase 3/7 activation. These findings pave the way for the possible use of these molecules as potential anticancer agents or as precursors for the generation of new and more potent and selective compounds against tumor cells. Microalgae are an excellent source of valuable compounds for nutraceutical and cosmeceutical applications. These photosynthesizing microorganisms are amenable for large-scale production, thus overcoming the bottleneck of biomass supply for chemical and activity characterization of bioactive compounds. This characteristic has recently also prompted the screening of microalgae for potential pharmaceutical applications. Here, we show that monoacylglycerides (MAGs) purified from the marine diatom  Chlorella ovalis and the dinoflagellate Amphidinium carterae suppressed the growth of HL-60 cells (human promyelocytic leukemia cell line) +.1-mono-


The use of cell lines in research can be affected by cell line misidentification. Short tandem repeat (STR) analysis is an effective method, and the gold standard, for the identification of the genetic origin of a cell line, but methods that allow the discrimination between cell lines of the same genetic origin are lacking. Here, we use intact cell MALDI-ToF mass spectrometry analysis, routinely used for the identification of bacteria in clinical diagnostic procedures, for the authentication of a set of cell lines consisting of three parental neuroblastoma cell lines  and eleven drug-adapted sublines. Principal component analysis (PCA) of intact-cell MALDI-ToF mass spectrometry data revealed clear differences between most, but not all, of the investigated cell lines. Mass spectrometry whole-cell fingerprints enabled the separation of IMR-32 and its clonal subline IMR-5. Sublines that had been adapted to closely related drugs, for example, the cisplatin- and oxaliplatin-resistant UKF-NB-3 sublines and the vincristine- and vinblastine-adapted IMR-5 sublines, also displayed clearly distinctive patterns. In conclusion, intact whole-cell MALDI-ToF mass spectrometry has the potential to be further developed into an authentication method for mammalian cells of a common genetic origin. Cell line misidentification has been a major issue since the first mammalian cell lines were established ,2. HeLa,While there are established methods for the determination of the genetic origin of cell lines, methods for the discrimination of cell lines derived from the same genetic origin (isogenic cell lines), such as clonal sublines, different cell lines derived from the same organism, or sub-cell lines that were adapted to growth under specific conditions are currently under development. Intact-cell MALDI-ToF mass spectrometry is routinely used for the identification of bacteria in clinical diagnostic procedures  and has One group of cell lines where STR methods are not able to distinguish between them is drug-adapted cancer cell lines, which are used as in vitro models of acquired drug resistance. The ATP-binding cassette (ABC) transporters ABCB1  and ABCC1 , major mediators of drug resistance in cancer, were discovered in drug-adapted cell lines ,32. More5 cells [17 in Caucasian- and Hispanic-American populations. The genetic origin of all cell lines was successfully confirmed  could discriminate between different cell lines. First, we looked at the parental cell lines IMR-32, IMR-5 and UKF-NB-3 using the comparisons PC1 vs. PC2, PC1 vs. PC3 and PC2 vs. PC3. The comparison PC2 vs. PC3 showed the largest separation . NotablyrCDDP1000) and oxaliplatin (UKF-NB-3rOXALI2000). It further separated the two sublines that had been adapted to closely related platinum drugs , topotecan (IMR-32rTOPO8) and melphalan (IMR-32rMEL500) displayed clearly distinctive patterns. Only the oxaliplatin-resistant IMR-32 subline (IMR-32rOXALI800) was difficult to differentiate from the parental IMR-32 cells  and the cisplatin-adapted (IMR-5rCDDP1000) subline formed clearly distinctive clusters (rDOCE20), dacarbazine-resistant (IMR-5rDACARB40) and vincristine-resistant (IMR-5rVCR10) sublines were harder to distinguish, although they also formed individual clusters  did not cluster closely together with regard to their mass spectrometry profiles. Further research will have to show whether this observation has functional implications.When directly comparing the STR and intact-cell MALDI-ToF analysis, as stated above, the STR approach showed four of the eleven drug-adapted sublines displayed identical STR profiles to the respective parental cell lines and the other sublines showed one to four changes, and hence all sublines displayed a >80% match with the respective parental cell lines, regarded as the threshold for authenticity  and hencIn conclusion, intact-cell MALDI-ToF mass spectrometry is a promising technique for the authentication of mammalian cells of a common genetic origin, but further development is required before it can be used as a reliable, generally applicable method, either on its own or (which seems to be more likely for the foreseeable future) in combination with other established methods, in particular STR typing. This approach represents a rapid method of authenticating cell lines that could complement traditional STR profiling. In developing this approach to authentication, we recommend that a minimum of three replicate analyses be undertaken for each sample to account for technical variation, and ideally three biological samples would also be analysed. We also suggest that a bank of quality controls consisting of historical fingerprints of authenticated cell lines be curated alongside frozen samples of authenticated cells that could be analysed alongside naive samples to account for any day-to-day variation in analyses and to give further confidence to identification of unknown cell lines. Further research will have to (1) establish that the methodology is able to identify unknown cell lines using statistically robust validation approaches and by masking the identification of cell lines to experimentalists, (2) show the extent to which cross-contaminations can be detected based on mass spectrometry profiles, (3) investigate the potential influence of cell culture conditions on mass spectrometry profiles and (4) establish databases with cell line profiles and quality standards for wider application by the community. Notably, methods for the authentication of cell lines of the same origin will also be important for areas beyond cancer research. For example, mouse strains are often derived from inbred strains and can also not be distinguished by STR ,59.The MYCN-amplified neuroblastoma cell line UKF-NB-3 was established from a bone marrow metastasis derived from a stage 4 neuroblastoma patient . The MYCrCDDP1000) or oxaliplatin (UKF-NB-3rOXALI2000), IMR-32 sublines adapted to carboplatin (IMR-32rCARBO1000), melphalan (IMR-32rMEL500), oxaliplatin (IMR-32rOXALI800) or topotecan (IMR-32rTOPO8), and IMR-5 sublines adapted to cisplatin (IMR-5rCDDP1000), dacarbazine (IMR-5rDACARB40), docetaxel (IMR-5rDOCE20), vinblastine (IMR-5rVINB20) or vincristine (IMR-5rVCR10) were established by continuous exposure to stepwise increasing drug concentrations, as previously described [https://research.kent.ac.uk/ibc/the-resistant-cancer-cell-line-rccl-collection/).UKF-NB-3 sublines with acquired resistance to cisplatin  supplemented with 10% foetal calf serum (FCS), 100 IU/mL penicillin and 100 µg/mL streptomycin at 37 °C. Cells were routinely tested for mycoplasma contamination.Cell lines were authenticated with the current STR typing technique of the DSMZ , which, according to the STR Typing Standard (ASN-0002: Human cell line authentication: STR profile standardization), uses an expanded set of STR markers that goes beyond the standard set of eight markers .Amplification was performed in DNase- and RNase-free vials of 0.2 mL in an i-Cycler . Per sample, the master mix contained 10 pmol total primer solution (1 µL of a 10 µM solution), 2.5 µL 10 x hot start PCR buffer (any supplier), 1 µL dNTP solution (5 µM), 0.2 µL hot start Taq polymerase solution , 19.5 µL distilled water and 1 µL genomic DNA solution (10 ng/µL). The genomic DNA was added last and independent from the master mix. The PCR programme was carried out as follows: 95 °C for 3 min, 1 repeat; 94 °C for 30 s, 57 °C for 30 s, 72 °C for 45 s, 30 repeats; 60 °C for 15 min, 1 repeat.For fragment detection and generation of allelic STR lists, aliquots of 1 µL of PCR products were combined with 0.25 µL of an internal size standard  in a total volume of 30 µL of sample loading solution on a microtiter plate. The samples were loaded automatically and analysed by the capillary electrophoresis system CEQ 8000 (Beckman-Coulter) using established fragment analysis parameters. Generated STR profiles were submitted to search within a reference STR database of cell lines . Additio4 viable cells were transferred to a 96-well plate format rack, centrifuged for 5 min at 960 rcf, and the supernatant removed. After washing by resuspension and centrifugation in 0.5 mL of PBS and in 0.35 M sucrose, cell pellets were stored at –80 °C until analysed.The intact-cell MALDI-ToF mass spectrometry analysis was performed essentially as previously described . 6.25 × 20 mg/mL (saturated) solution of sinapinic acid was dissolved in matrix buffer ) and sonicated in a water bath for 15 min before centrifugation. After removal from –80 °C storage, cells were allowed to equilibrate to room temperature for 15 min prior to resuspension in 50 µl of sinapinic acid solution and incubation at 4 °C for 3 h. Then, 1 µl of each sample was spotted onto a 384 MTP ground steel MALDI-ToF plate  and air-dried, before the plate was placed into the MALDI-ToF mass spectrometry instrument (Bruker Ultraflex).n = 3) biological cultures were analysed, each culture analysed by triplicate technical analyses of the samples.Spectra were collected using the following MALDI-ToF ms instrument settings: laser frequency: 20 Hz; polarity +ve; ion sources: 1. 20 kV, 2. 17.25 kV, lens 5.0 kV; gating mode: maximum strength; suppress @ 4000 Da; pulsed ion extraction 550 nS; range 5000–60,000 Da; sample rate 0.1 Gs/s; resolution enhanced 100 mV electronic gain; smooth high. For each sample, 100 shots were summed and saved. In all cases, triplicate (m/z vector); 2. Baseline Correction (removes the effect of noise introduced by the matrix); 3. Filtering ; 4. Alignment ; 5. Quality Control ; 6. Normalisation . Details for all steps have been outlined previously in [http://tinyurl.com/msresample). In addition, a baseline correction was performed using the “msbackadj” function in the MATLAB Bioinformatics Toolbox (http://tinyurl.com/msbackadj). Peak alignment was performed using the “msalign” function from the MATLAB Bioinformatics Toolbox . Finally, data was normalised using the “msnorm” function from the MATLAB Bioinformatics Toolbox , as described in [Data files were exported from the Bruker Flex Analysis software in ASCII format for preprocessing in MATLAB (Version R2017a for Windows) prior to the application of the principal component analysis as described in . The folously in . Resamplribed in .


Dasatinib (Sprycel) was developed as a tyrosine kinase inhibitor targeting Bcr-Abl and the family of Src kinases. Dasatinib is commonly used for the treatment of acute lymphoblastic and chronic myelogenous leukemia. Previous clinical studies in melanoma returned inconclusive results and suggested that patients respond highly heterogeneously to dasatinib as single agent or in combination with standard-of-care chemotherapeutic dacarbazine. Reliable biomarkers to predict dasatinib responsiveness in melanoma have not yet been developed.Here, we collected comprehensive in vitro data from experimentally well-controlled conditions to study the effect of dasatinib, alone and in combination with dacarbazine, on cell proliferation and cell survival. Sixteen treatment conditions, covering therapeutically relevant concentrations ranges of both drugs, were tested in 12 melanoma cell lines with diverse mutational backgrounds. Melanoma cell lines responded heterogeneously and, importantly, dasatinib and dacarbazine did not synergize in suppressing proliferation or inducing cell death. Since dasatinib is a promiscuous kinase inhibitor, possibly affecting multiple disease-relevant pathways, we also determined if basal phospho-protein amounts and treatment-induced changes in phospho-protein levels are indicative of dasatinib responsiveness. We found that treatment-induced de-phosphorylation of p53 correlates with dasatinib responsiveness in malignant melanoma.Loss of p53 phosphorylation might be an interesting candidate for a kinetic marker of dasatinib responsiveness in melanoma, pending more comprehensive validation in future studies. Dasatinib (N-(2-chloro-6-methyl-phenyl)-2-((6-(4-(2-hydroxyethyl)-piperazin-1-yl)-2-methylpyrimidin-4-yl)amino)-1,3-thiazole-5-carboxamide) is an orally active tyrosine kinase inhibitor developed to target Bcr-Abl and the family of Src kinases . LargelyDasatinib has been and still is being tested as a single agent and in combination therapies, for example together with other kinase inhibitors or genotoxic chemotherapeutics, in haematological cancers and solid tumours. At the time of writing, clinicaltrials.gov lists 72 active and planned studies. In melanoma, the therapeutic efficacy of dasatinib was tested as a single treatment as well as in combination with the standard-of-care chemotherapeutic dacarbazine (DTIC) in phase I/II trials. In a phase I trial with 29 patients, the reported objective response rate (ORR) was 13.8%, and the 6-month progression-free survival (PFS) was 20.7%. In a phase II trial with 36 patients, the ORR was 5%, and the 6-month PFS was 13%. . With thDasatinib and dacarbazine both were reported to impair cell proliferation, either by kinase inhibition or by DNA damage-induced cell cycle arrest , 12. To 72 h after treatment commenced, heterogeneous responses to dasatinib or dacarbazine on cell proliferation were observed. For dasatinib single agent treatment, WM115 was the most sensitive cell line, with 10–100 nM sufficient to reduce proliferation to approximately 25% ), a target associated with c-Src signaling, directly affected by dasatinib and also known for being implicated in downstream RAS/RAF/MEK/ERK signaling, thereby affecting cell growth and proliferation [97), a taBasal phospho-protein expression across the melanoma cell line panel was highly heterogeneous Fig. a. ComparHere, we studied the responsiveness of melanoma cells to dasatinib, alone or in combination with standard-of-care chemotherapeutic dacarbazine, by systematically analyzing growth inhibition and cell death at therapeutically meaningful concentrations ranges of both drugs. To our knowledge, this is the most comprehensive quantitative analysis of the effect of these therapeutics on melanoma cell proliferation and viability to date. Melanoma cells responded heterogeneously, with both dasatinib-responsive and highly dasatinib-resistant cell lines identified. Of thirteen phosphoproteins studied, all of which are directly or indirectly involved in regulating cell growth, proliferation, or cell cycle progression, none correlated with dasatinib responsiveness in their expression amounts. Interestingly though, the amounts of phosphorylated p53 (S46) dropped significantly in dasatinib-responsive cell lines following treatment, highlighting this response as a previously unknown kinetic marker candidate for susceptibility to dasatinib. Determining the specificity of such a marker more accurately, however, would require further studies, since p53 phosphorylation also dropped in one non-responsive cell line (WM3060).p = 0.06), indicating that modulators of Src activity might provide scope for marker development [Dasatinib has been studied comprehensively for its spectrum of kinase targets. Dasatinib inhibits diverse kinases besides the Src family at therapeutically meaningful concentration ranges. These targets include FAK, BTK, EPHs and p38 MAPK and others . It therelopment . c-KIT, elopment , can be elopment , 10, 32.Instead of studying pre-treatment markers, an alternative approach lies within investigating if kinetic markers for dasatinib responsiveness can be identified. We found that the amounts of p53 (S46) drop upon dasatinib treatment in responsive melanoma cell lines. Among other kinases, the direct dasatinib target p38 MAPK can phosphorylate p53 at S46 . p53 is w/v) heat inactivated fetal bovine serum (Sigma-Aldrich), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich). Cells were grown at 5% CO2 and 37 °C. To study treatment responses, cells were seeded into flat bottom 96 well plates and treated with dasatinib , dacarbazine  or combinations of both drugs. Drugs were added simultaneously.Cells were obtained as authenticated stocks from the ATCC  , the Wistar Institute   and the DSMZ  (Mel Juso). Five cell lines carried activating BRAF mutations , four cell lines NRAS mutations , one cell line a c-KIT mutation (WM3211), and two cell lines were BRAF/NRAS/c-KIT wildtype . Cells were cultured in DMEM  supplemented with 4 mM L-glutamine, 4.5 g/l glucose, 10% . Measurements were performed on a BD LSRII SORP HTS cytometer . Data files were exported in .fcs format and analyzed using Flowing Software . All experiments were independently repeated at least three times and all treatment conditions were measured in triplicates.Cells were seeded into 96 well plates and lysed after 24 h treatment with dasatinib. The protein concentrations in cell lysates were adjusted to 300 μg/ml, using the BCA assay kit for protein concentration determination (Pierce). xMAP bead-based ELISA type assays  were performed on a Luminex FLEXMAP 3D® instrument using a custom developed phosphoprotein 13-plex panel : RAC-alpha serine/threonine-protein kinase (AKT) - S473, Focal adhesion kinase 1 (FAK1) - Y397, Transcription factor AP-1 (c-JUN) - S63, dual specificity mitogen-activated protein kinase kinase-1 (MEK1/MP2K1) - S217/S221, Mitogen-activated protein kinase-3 (ERK1/MK03) - T202/Y204, Mitogen-activated protein kinase 9 (JNK2/MK09) - T202/Y204, Tyrosine-protein phosphatase non-receptor type 11 (PTN11/SHP2) - Y542, Cyclic AMP-responsive element-binding protein 1 (CREB) - S133, NF-κB inhibitor alpha (IκBa) - S32/S36, Ribosomal protein S6 kinase beta-1 (KS6B1/P70S6K) - T389, Cellular tumor antigen p53 (P53) - S46, Signal transducer and activator of transcription 3 (STAT3) - Y705, Serine/threonine-protein kinase WNK1 (WNK) - T60. For normalization, the levels of total GAPDH protein were analyzed.Melanoma cell lines were homogenized in lysis buffer  with addition of PhosSTOP phosphatase inhibitor cocktail and cOmplete protease inhibitor cocktail . Total protein concentrations of samples were determined by Bradford protein assay . Samples were loaded (30 μg) and separated on Bolt 4–12% Bis-Tris Plus Gels  at 150 V, 400 mA for 40 min. Proteins were transferred to nitrocellulose membranes using the iBlot 2 gel transfer device . The membranes were blocked in 5% BSA in TBST for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used: rabbit-anti p53 , anti-mouse beta-Actin  and rabbit-anti phospho-p53 (S15) , anti-rabbit p38 MAPK , anti-mouse phospho-p38 MAPK (T180 / Y182) , anti-mouse alpha-Tubulin ,. The membranes were washed 3 × 10 min in TBST and incubated with secondary antibody HRP goat-anti rabbit  for 1 h on a room temperature followed by 3 × 10 min wash in TBST. Luminata Forte Western HRP substrate  was added and chemiluminescence signal was detected on Amersham Imager 600 system . Signal intensities above background were quantified using ImageJ software.GraphPad Prism  was used for statistical analysis.