Almost all living cells maintain size uniformity through successive divisions. Proteins that over and underscale with size can act as rheostats, which regulate cell cycle progression. Using a multiomic strategy, we leveraged the heterogeneity of melanoma cell lines to identify peptides, transcripts, and phosphorylation events that differentially scale with cell size. Subscaling proteins are enriched in regulators of the DNA damage response and cell cycle progression, whereas super-scaling proteins included regulators of the cytoskeleton, extracellular matrix, and inflammatory response. Mathematical modeling suggested that decoupling growth and proliferative signaling may facilitate cell cycle entry over senescence in large cells when mitogenic signaling is decreased. Regression analysis reveals that up-regulation of TP53 or CDKN1A/p21CIP1 is characteristic of proliferative cancer cells with senescent-like sizes/proteomes. This study provides one of the first demonstrations of size-scaling phenomena in cancer and how morphology influences the chemistry of the cell. Molecules that super- and subscale with cancer cell size and the consequences for cell growth are defined. Xenopus oocytes  (P < 0.001). By retrieving the W statistic, we calculated the “common language effect size” for each comparison. This produced a matrix of pairwise comparisons between all cell lines that measured the degree of difference in median area between them. Clustering the lines according to this difference, let us define three area classes , and the CV differed only modestly between cell lines   . This su2) and 5555 (490 μm2)] and large [B14341 (1500 μm2) and 17864A (900 μm2)] cell lines are largely 2N, and all exhibit partial 4N populations. We note that 21917 (800 μm2) and 24038 (2400 μm2) were almost entirely tetraploid.Previous studies have indicated that DNA content  when populations are pooled and  when considered independently  and identified two distinct clusters of cell lines: a set of smaller cell lines with a relatively high D/C and a set of large lines with a relatively low D/C ratio  across lines. This metric differs from ploidy because it considers the amount of Hoechst staining within the nucleus, which can be affected by factors such as packing, but facilitates the measurement of many more cells. We observed two independent linear relationships between DNA content and cell area across cell lines . Peptide expressions, normalized to reflect the relative difference in mass fractions across cells lines (Methods), were correlated to cell areas revealing proteins whose concentrations continuously scale with size.RB1 concentration at which cells commit to division , P < 0.05, n = 10 (or 11)] ] . SpecifiR = −0.38, P > 0.05, n = 11)  pathway, rather than across sizes (see data S2 for phosphoproteomic data on the MAPK pathway in these cell lines). Despite their size and low RB1 mass fraction, 21015 BRAF and C876 NRAS exhibited a CCND1 mass fraction comparable to smaller cell lines, whereas our similarly large KDBRAF cell lines, 24038 and B14341, showed the lowest CCND1 expression   (data S2). We concluded that the set point, where CDK4/6:CCND1 activation exceeds RB1 activity to putatively drive proliferation, is thus similar across lines.CCND1 abundance was found to be weakly negatively correlated with size ( n = 11) . This su = 2, 9) . The ratCCND1, we established a method to quantify the signaling activity upstream of CCND1 using the phosphorylation state of transcriptional regulators of CCND1, as defined by the ENCODE database ( CCND1regs) across different melanoma lines. All phosphorylations used in the analysis with known causative kinases or documented cellular effects . Moreover, this implies that the CCND1 levels in larger lines are not due to differences in mitogen signaling alone.We observed a negative relationship between cell size and the ratio between RB1/pCCND1reg ratio  is associated with lower expression of ribosomal and spliceosome proteins   .R| >0.55), which were then sorted into two groups. One group of peptides are those expressed in class 1/2  cells  and those expressed in class 2/3 (large) cells . We conducted an ontological analysis on all the hit peptides (P <10−9). These proteins included checkpoint mediators ATM, BRCA1, and WEE1 and the mitotic cyclin CCNB2. In contrast, class 2/3 large lines were statistically enriched for superscaling peptides from lipid/glycolipid metabolic processes and components of the ECM. These included MVK, MVD, ACAT2, and COL2A1   and biosynthetic processes , including the canonical mTORC1-activating phosphorylation, S2448 , implying up-regulation in small cell lines. Class 2/3 larger cell lines were enriched for superscaling phosphorylations on guanosine triphosphatase (GTPase) and cytoskeletal regulators  and also set of growth regulators , implying differential regulation of biosynthesis across the size range  and a CTNNB1 degradation signal (S29) , including BRCA1 S686 an AKT1 target and stabilizing phosphorylation (eEIF4B and EIF5B), “spliceosome machinery” , “cell adhesion” peptides , and increased expression of “growth signaling”  peptides (P < 10−3). Despite mTOR S2448 subscaling, many phosphorylations enriched in larger cell lines correspond to insulin signaling events such as EIF4B S497 .peptides   mass fractions and increased relative expression of ECM and lipid metabolic components. Further, large sizes coincide with increased phosphorylation of cytoskeletal effectors and differential regulation of the biosynthetic machinery.Larger cell sizes, related to lower D/C ratios, are associated with decreased G BARD1, WEE1, E2F8, and RB1) and control of gene expression  subscaled with cell size (enrichments significant to at least P < 1 × 10−3) , interferon induced protein with tetratricopeptide repeats 1, interferon regulatory factor 5 (IRF5) and IRF7, and adenosine deaminase RNA specific] were up-regulated in larger cell lines (enrichments significant to at least P < 1 × 10−3).We next performed transcriptomic experiments to gain further insight into the relationship between size and signaling network organization and activity. We measured the abundance of 24988 RNA molecules overlapping with 9290 measured peptides (data S5). We conducted a volcano analysis, as described previously (Methods), to identify a list of transcripts that super- and subscale with size across lines . mRNA’s 2-M regulators, altered metabolism and biosynthesis, and an increased inflammatory (transcript) mass fraction.In conjunction with that observed in the proteomic data, these data show that compared to class 1/2 smaller cells, class 2/3 larger cells have decreased levels Gn = 9290), in agreement with previous studies  correlations with peptide abundances, we observed an obvious enrichment of cell cycle and DNA repair/replication genes   . As pept2), and recalculated the correlation coefficients between mRNA and peptide fractions. At the gene level, in the smaller subset, we observed enrichment of inflammatory, adhesion and cell cycle regulators  . In large cells, there was a strong correlation between mRNA and the protein mass fraction of ribosomal and translational genes     in the lnd ETF1) . This sund ETF1)  to the mTogether, these data suggest that the disruption of cell transcription relates to the subscaling of the biosynthetic mass fraction in larger, low D/C, cell lines. This may occur through the decreased RNAP expression at larger sizes. Depressed biosynthetic expression and signaling may underpin further scaling relationships.P < 10−3, e.g., “BOK,” “Toll-like receptor 3 (TLR3),” “TLR4,” “BCL2,” and “TICAM1”] and lipid/carbohydrate metabolism  is associated with large cell lines. Excluding the “overexpressed” genes, we observe enrichment of lipid metabolic proteins alone . In contrast, we observe a clear enrichment of cell cycle , mitotic  and DNA repair  peptides in smaller lines indicating subscaling (fig. S4 and data S3).To assess the universality of our size-scaling relationships, we extended our panel of 11 lines to include a further 12 composed of the same genotypes as before and conducted further quantitative morphological and phosphoproteomic experiments and analyses . In contrast to the previous analysis, we note an additional “arm” of the volcano plot indicating a subset of peptides extremely enriched in larger cell lines (fig. S3). We hypothesize that this represents gene overexpression rather than superscaling relationships, as there is no smooth transition in between the arms. Initially including these genes in the analysis, we found that an increased mass fraction of apoptotic effectors . A summWe noted that the α  and β (growth rate) parameters of our model showed nonlinear relationships with cell size ; thus, we expected that peptides whose levels correlated with α and β would be (partly) independent of those that correlate with size as determined by imaging . Thus, bR = 0.98). Fivefold cross validation was used to select the number of components and maximize transferability of the model  model predicting parameter values from the proteomic expression data  [false discovery rate (FDR) < 0.05], including d CDC25A . Expressand TP53 . Many ofTP53 and CDKN1A/p21, which covaried with proteins that are predictive of growth . However, these lines were notably different in their levels of CDNK1A and TP53. One group of BRAFKD cells has high growth rates and large size and is CDKN1A low and TP53 high. However, the other group of NRAS and BRAF mutant cells has high growth rates and large size and is CDKN1A high and TP53 low. Further, CDKN1A and CCND1 mass fractions were observed to scale linearly with one another . Thus, in these cell lines, a high growth rate (and large size) is associated with two distinct states: a /TP53low/p21high/CCND1high state and a /TP53high, /p21low/CCND1low state  dilution to size determination and control  stage cycle, implying the dominance of a small subset of proliferative regulators. This suggests a more central role for the RB1 subscaling observed in these cell lines.We constructed a simple theoretical model to demonstrate how continued growth under proliferative stress could maintain the cell proliferation rate. This relied on the probability of a cell transitioning to the next stage of the cell cycle being proportional to its size . This shows that larger cell lines maintain high growth rates despite down-regulation of anabolic pathways and decreased ribosomal mass fractions, qualities typically associated with decreased biosynthesis  and β (growth rate) parameters of our model, we found that they were best predicted by the expression of TP53 or CDKN1A/p21 may actually facilitate progression in large cells, by resolving stresses or acting at checkpoint proteins. For example, TP53 or CDNK1A/p21 expression in large cells might act to slow proliferation transiently (as cells grow), allowing engagement of the DDR  followed by centrifugation  and resuspension in complete medium. Cell counting was performed using a Countess automated cell counter with trypan blue exclusion (Thermo Fisher Scientific).Cell lines were maintained in standard culture conditions . Cells were incubated in DMEM media with 10% FBS and Primocin antibiotic at 37°C and 5% carbon dioxide. Cells were imaged at 4-hour intervals using the Incucyte imaging system. Nine fields of view were imaged from each well. Images were segmented using Ilastik image segmentation software to identify individual cells. The number of cells in each field of view was calculated using CellProfiler. Growth curves were plotted using the ggplot2 library from the R programming language. Average growth rates were computed as Gr = 2/3*(Mean_Area)/(Doubling_time), where doubling time is the average time taken for cell number to double in the population.Samples were fixed in freshly prepared 4% paraformaldehyde (PFA)/phosphate-buffered saline (PBS) for 15 min. Slides were subsequently permeabilized with 0.25% Triton/PBS for 10 min and blocked with 0.5% bovine serum albumin (BSA)/0.02% glycine/PBS for 30 min. Primary antibodies were introduced via the same solution in a 1:1000 dilution and left on for 1 hour. The slides were washed with PBS, and the same was carried out for the secondary antibodies (kept in the dark to avoid bleaching). Hoechst stain was added postsecondary (1:500) to stain DNA as was phalloidin to stain actin.Image acquisition was performed using an Opera Cell:Explorer-automated spinning disk confocal microscope. Twenty fields of view were imaged in each well . Cell segmentation was performed using Acapella software (PerkinElmer). Nuclei were segmented using the Hoechst channel (405 to 450) and cell bodies defined by the tubulin signal (568 to 603). Geometric features measured include the area of all subcellular regions; the length, width, and elongation (length/width) of the cell and nucleus; cell and nuclear roundness; and nucleus area/cytoplasm area.Statistical tests were carried out in the MATLAB (MathWorks) environment. Cell area data were “acosh”-transformed to induce a normal distribution of areas in each cell line and standardize the variances before ANOVA and Mann-Whitney/Wilcoxon tests. Standardization success was determined using the Shapiro-Wilks normalization test, ensuring that the data are normally distributed, and the Bartlett test, to guarantee equal variances across lines.6 cells were seeded in Falcon T25 flasks and incubated overnight in media containing the appropriate aphidicolin concentration  before FACS analysis of DNA content as above.Cells were trypsinized and harvested into a 15-ml falcon tube for cell counting. After centrifuging the falcon at 2400 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1-ml of 1% fetal calf serum in PBS. Three-milliliter ice cold 100% ethanol was added dropwise to the cells while slowly vortexing and left to fix overnight. The cells were then pelleted by centrifugation for 5 min at 2400 rpm and resuspended in 5 ml of PBS. They were incubated at room temperature for 20 min. After centrifuging for 7 min at 1200 rpm, the pellet was resuspended in 1 ml of propidium iodide (PI) solution through the cell strainer into a FACS tube. The PI solution was made with 1:100 PI at 5 mg/ml and 1:1000 ribonuclease A at 10 mg/ml in PBS. The cell cycle composition was measured using the BDSAria, and the data were analyzed using FlowJo. For EdU (5-ethynyl-2′-deoxyuridine) incorporation assays, cells were treated with a final concentration of 10 μM EdU before harvesting. Instead of fixing with ethanol and staining with PI, cells were resuspended in 4% PFA for 15 min at room temperature. They were then pelleted by centrifugation and PFA aspirated, followed by a wash. Five hundred microliters of the appropriate Thermo Fisher Scientific Click-iT reaction cocktail was added to each sample and incubated for 30 min in the dark, according to the manufacturer’s instructions. Cells were washed once, stained, and then transferred via a cell strainer into a FACS tube for analysis as above. Washes used 1% BSA in PBS. Staining used Hoechst (20 μg/ml) added to 0.1% Triton-X in PBS. If applicable, 10Cell pellets were dissolved in 150 μl of lysis buffer containing 1% sodium deoxycholate, 100 mM triethylammonium bicarbonate, 10% isopropanol, 50 mM NaCl, and Halt protease and phosphatase inhibitor cocktail (100×)  on ice with pulsed probe sonication for 15 s. Samples were boiled at 90°C for 5 min and sonicated for another 5 s. Protein concentration was measured with the Quick Start Bradford Protein Assay (Bio-Rad) according to the manufacturer’s instructions. Aliquots containing 100 μg of protein were reduced with 5 mM tris-2-carboxyethyl phosphine for 1 hour at 60°C and alkylated with 10 mM iodoacetamide for 30 min in the dark. Proteins were then digested overnight by adding trypsin at final concentration of 75 ng/μl (Pierce). The resultant peptides were labeled with the tandem mass tag (TMT)-11plex reagents (Thermo Fisher Scientific) according to the manufacturer’s instructions and were combined in equal amounts into a single tube. The combined sample was then dried with a centrifugal vacuum concentrator. Two technical replicate TMT batches from the same protein extracts were prepared to assess reproducibility. One TMT batch was fractionated offline with high-pH reversed-phase chromatography using the XBridge C18 column  on a Dionex UltiMate 3000 high-performance liquid chromatography (HPLC) system. Mobile phase A was 0.1% ammonium hydroxide (v/v), and mobile phase B was acetonitrile and 0.1% ammonium hydroxide (v/v). The TMT-labeled peptide mixture was reconstituted in 100 μl of mobile phase A and fractionated with a gradient elution method at 0.2 ml/min as follows: for 5-min isocratic at 5% B, for 35-min gradient to 35% B, gradient to 80% B in 5 min, isocratic for 5 min, and re-equilibration to 5% B. Fractions were collected every 42 s and vacuum-dried. The second TMT replicate batch was fractionated with the Pierce High pH Reversed-Phase Peptide Fractionation Kit according to the manufacturer’s instructions.Peptide fractions from the first TMT batch were reconstituted in 10 μl of 20% isopropanol and 0.5% formic acid binding solution and were loaded on 10 μl of phosphopeptide enrichment immobilized metal affinity chromatography resin  already washed and conditioned with binding solution in custom-made filter tips fitted on Eppendorf tube caps. After 2 hours of binding at room temperature, the resin was washed three times with 40 μl of binding solution at 300 g, and the flow-through solutions were collected for total proteome analysis. Phosphopeptides were eluted three times with 70 μl of 40% acetonitrile and 400 mM ammonium hydroxide solution. Eluents and flow-through samples were vacuum-dried and stored at −20°C until the liquid chromatography–mass spectrometry (LC-MS) analysis.5, and 50 ms ion trap (IT) with the top speed mode in 3 s and were isolated for collision-induced dissociation (CID) fragmentation with quadrupole isolation width 0.7 Th. Collision energy (CE) was 35% with AGC 1 × 104 and IT 50 ms. MS3 quantification was obtained with Higher-energy C-trap dissociation (HCD) fragmentation of the top five most abundant CID fragments isolated with Synchronous Precursor Selection. Quadrupole isolation width was 0.7 Th, CE 65%, AGC 1 × 105, and 105 ms IT. The HCD MS3 spectra were acquired for the mass range 100 to 500 with 50 K resolution. Targeted precursors were dynamically excluded for further isolation and activation for 45 s with 7 parts per million (ppm) mass tolerance. Phosphopeptide samples were analyzed with an HCD method at the MS2 level with CE 38%, AGC 1 × 105 and max IT 105 ms.LC-MS analysis was performed on the Dionex UltiMate UHPLC 3000 system coupled with the Orbitrap Lumos mass spectrometer (Thermo Fisher Scientific). Peptides were loaded to the Acclaim PepMap 100, 100 μm by 2 cm C18, 5 μm, 100 Ȧ trapping column at a flow rate of 10 μl/min. The sample was then subjected to a gradient elution on the Acclaim PepMap RSLC  C18 capillary column at 45°C. Mobile phase A was 0.1% formic acid, and mobile phase B was 80% acetonitrile and 0.1% formic acid. The separation method at flow rate 300 nl/min was as follows: for 90 min (or 150 min for the replicate batch) gradient from 10 to 38% B, for 10 min up to 95% B, for 5 min isocratic at 95% B, reequilibration to 10% B in 5 min, for 10 min isocratic at 10% B. Precursors between 375 and 1500 mass/charge ratio were selected with mass resolution of 120 K, automatic gain control (AGC) 4 × 10q value and decoy database search. Spectra were searched against reviewed UniProt mouse protein entries. The reporter ion quantifier node included a TMT 11plex quantification method with an integration window tolerance of 15 ppm and integration method based on the most confident centroid peak at the MS3 or MS2 level. Only unique peptides were used for quantification, considering protein groups for peptide uniqueness. Peptides with average reporter signal-to-noise of >3 were used for quantification.The SequestHT search engine was used to analyze the acquired mass spectra in Proteome Discoverer 2.2 (Thermo Fisher Scientific) for protein identification and quantification. Precursor mass tolerance was 20 ppm, and fragment ion mass tolerance was 0.5 Da for the CID and 0.02 Da for the HCD spectra. Spectra were searched for fully tryptic peptides with maximum of two miss-cleavages. TMT6plex at N terminus/K and carbamidomethyl at C were defined as static modifications. Dynamic modifications included oxidation of M and deamidation of N/Q. Dynamic phosphorylation of S/T/Y was included for the phospho-enriched samples. Peptide confidence was estimated with the percolator node. Peptide FDR was set at 0.01, and validation was based on R, and a fold change was calculated between cells lying above or below the mean. Peptides scoring Fc > 1.5 or <0.66 and |R| > 0.55 were taken as hit super/subscaling peptides. The Fc threshold of 1.5 stems from a power analysis: An Fc of 1.5 is the minimal detectable difference between groups we can detect by a t test between small and large cell line clusters  when accepting a true-positive rate of 0.95 . The fold change was defined asA” denotes the mean abundance of the peptide in cluster 1, and M(A) denotes the minimal value of the mean of cluster 2 for there to be a detectable difference in mean as above. The “100” stems from all peptides being scaled across line to have a mean value of 100. This value was calculated using the “samplesizepwr” function in MATLAB (MathWorks) statistics and machine learning toolbox. Transcripts are treated identically to peptides.Peptide abundances were scaled relative to other detected peptides in the sample such that they reflect abundance/total protein mass. The expression of each peptide was correlated to average cell line area to derive a correlation coefficient, Geneontology.org’s downloadable database  according to the manufacturer’s protocol. The evaluation of the isolated RNA integrity and quantity was carried out by the Agilent TapeStation system using an RNA ScreenTape .For the mRNA library preparation, 4000 ng of total RNA was treated with TurboDNase to remove genomic DNA contamination, . PolyA RNA was selected from 1000 ng of the purified RNA using NEBNext mRNA magnetic isolation module  following the manufacturer’s directions. From the resulting mRNA, strand-specific libraries were created using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina . Final libraries were quantified using quantitative polymerase chain reaction and clustered at a molarity of 300 pM. Sequencing was performed on an Illumina NovaSeq 6000 (Illumina) using paired end ×100 cycles v1.0 chemistry, to achieve coverage of 25 million reads per sample.k/α (the expected mean of the distribution). Every generation, the area distribution is convolved with the mass-gain distribution, computed by performing an inverse Fourier transform on the product of the two distributions respective Fourier transforms. This produces the division area distribution, Ad(A), which must be transformed to Ad(2A) to capture the effects of cell division. We perform this by setting Ab(Ax) = Ad(Ai) + Ad(Ai + 1), where “I” = xn − xn-1 for all x, where Ab denotes the birth size distribution. This is then convolved with the gain distribution as before to generate the next division distribution and so on until a desired number of generations has been reached.The initial cell area distribution is considered a delta function centered on P = αAdiv, and a random number, “r,” is drawn from a flat distribution. Should r be less than the division probability of a cell, the cell divides symmetrically in two, adding a new cell to the population with half the size of the mother, and halving the mother size. If r is greater than the division probability, then the cell size increases according to β = kAdiv. This system continues until a final cell count of 20,000 was achieved.An initial population of 1000 cells was assigned an α and β value and a random initial area. At each time step, the division probability for each cell is calculated, according to Initially, α values were exhaustively tested (β is determined from the proliferation and area measurements on a per cell line basis). For each, we calculated the Kullbeck-Liebler divergence between the experimental and simulated dataHaving identified approximate minima from the low-resolution parameter screen, we used the values defining this region as an initial state for a stochastic gradient descent minimizing along the gradientModel fitting was conducted within the commercial MATLAB (MathWorks) software’s machine learning toolbox.r-squared metric.Regression analyses were conducted with the MATLAB (MathWorks) environment using the plsregress function from the machine learning toolbox. Partial least square regression was selected as the method to help mitigate the influence of colinearity in the predictor dataset. Model components were selected through fivefold cross validation using the elbow method on the mean square error as a function of component number. All peptides were mean-centralized before model construction. Fit quality was assessed through the wjf is the weight value for j variable and f component, and SSYf is the sum of squares of explained variance for the fth component and J number of X variables. SSYtotal is the total sum of squares explained of the dependent variable, and F is the total number of components. Features with a VIP score greater than 1 were taken as major drivers of the model. VIP scores of peptides with negative weights in the first PLSR component were made negative in preparation for enrichment analysis.The influence a feature has on a model was estimated through VIP scores calculated asGSEA was conducted using the “WebGestalt” web application on our ranked list of peptides (VIP score defined the rank) (


Myrica cerifera root bark, was investigated using enzymatic activity assays and molecular docking procedures. Dimethylmyricacene was shown to inhibit both the cleavage and the religation steps of the enzymatic reaction, and cell viability of A-253, FaDu, MCF-7, HeLa and HCT-116 tumor cell lines.Human topoisomerase 1B regulates the topological state of supercoiled DNA enabling all fundamental cell processes. This enzyme, which is the unique molecular target of the natural anticancer compound camptothecin, acts by nicking one DNA strand and forming a transient protein–DNA covalent complex. The interaction of human topoisomerase 1B and dimethylmyricacene, a compound prepared semisynthetically from myricanol extracted from  Human DNA topoisomerase 1B (hTop1) is a ubiquitous enzyme, essential in mammals . This moSilybum marianum, that alone does not show any effect on hTop1, but inhibits the enzyme, in the form of oxidovanadium (IV) complexes [Natural products (NPs) remain an extensive and excellent source of inspiration for the development of new pharmaceuticals, and most of the drugs currently used in the clinic are NPs that display inhibition towards a specific target . Howeveromplexes . Anotheromplexes .Myrica cerifera root bark [Modification of these naturally occurring compounds led to the discovery of several hTop1 catalytic inhibitors, such as CYB-L10, that has a higher hTop1 inhibition activity, compared with its parent indolizinoquinolinedione . Identifoot bark . Reportsoot bark . Concernoot bark . In thisHTop1, for time course and dose dependent assays, has been expressed in the top1 null EKY3 yeast strain and purified by affinity chromatography, as described elsewhere . To valiDulbecco’s modified Eagle’s medium high glucose (DMEM), RPMI 1640 medium, fetal bovine serum (FBS), L-glutamine and penicillin/streptomycin, were purchased from Euroclone , McCoy’s and Minimum essential Medium Eagle (EMEM) were purchased from the Lonza group Ltd. . Complete media (CM) were supplemented with 10% FBS, 2 mM L-glutamine, 0.1 mg/mL streptomycin and 100 U/mL penicillin. The ovarian cancer cell line SKOV-3 was purchased from Cell Biolabs Inc., and maintained in DMEM-high glucose, CM.The colorectal adenocarcinoma cell line, HT-29, colorectal carcinoma cell line HCT-116 and melanoma cell SK-MEL-28, were gifted by Dr Cinzia Tesauro  and maintained in RPMI 1640 CM. A non-small-cell lung cancer cell line, A-549, triple negative breast cancer cell SUM-159, MDA-MB-468 and MDA-MB-231, HER2/c-erb-2 positive breast cancer cell line SK-BR-3, MCF-7 and T-47-D ER+, PR+/–, HER2– and the adenocarcinoma HeLa cell line were provided by Dr. Giuseppe Sconocchia  and were maintained in DMEM-high glucose, CM. The submandibular gland squamous cell carcinoma cell line A-253 and hypopharyngeal squamous cell carcinoma cell line FaDu were gently provided by Dr. Angelo Peschiaroli  and maintained in McCoy’s CM and E-MEM CM, respectively. All cell lines were kept in culture for a maximum of eight passages and were tested for mycoplasma, using the PCR detection Kit (Euroclone).1H NMR spectra of DM was recorded in CDCl3, using a Varian 600 MHz broadband instrument and is reported in DM was produced, according to reference . Briefly2 and 5 μg/mL acetylated bovine serum albumin) with 150 mM KCl, 0.5 μg pBlueScript and 1 U of purified htop1, with different concentrations of DM. As a positive control, the enzyme was incubated with the same amount of ACN used to dissolve the compound. Following 1 h incubation at 37 °C, the reaction was stopped by adding 0.5% SDS stop dye. To evaluate the reversibility or irreversibility of DM, the same experiment was carried out with 100 μM of DM as a function of time, and reactions were stopped at the indicated time points with a final concentration of SDS 0.5%. All samples were run on 1% agarose gel with TBE 1 X buffer . The presence of relaxed supercoiled DNA was visualized through a UV transilluminator after a gel staining in 0.5 μg/mL EtBr and destaining in dH2O.To assess the minimal dose of DM inhibiting hTop1 a dose dependent relaxation assay of negatively supercoiled DNA pBlueScript KSII (-) was performed. In a volume of 30 μL, were mixed TOPO mix buffer 1X . The cleavage reaction was performed at different time points with 100 μM of DM, by incubating 0.6 pmol of SS with 1.2 pmol hTop1 (Prospec), as previously described . The rea2 with the appropriate medium, to evaluate the cell viability, as previously described [2. Following the incubation, the medium was then removed and replaced with 0.5 mg/mL of MTT reagent in a final volume of 200 μL diluted with the medium. The plates were incubated again for 4 h at 37 °C, 5% CO2. Prior to measuring the absorbance at 570 nm, the medium was replaced with 100 μL of DMSO and placed on a shaking incubator for 15 min at room temperature, covered from light. The dose dependent assay cells were plated in a 96-well plate the day before the experiment. Then, 24 h later, the medium was removed and replaced with different amounts of DM. In detail, the cells were treated with 12.5, 25, 50 and 100 μM of DM and the same concentrations were used to treat cells with TPT as well, representing the positive cytotoxicity control. The plates were incubated again for 48 h at 37 °C under 5% CO2. To evaluate the minimal dose of DM reducing cell viability, the cells were subjected to the MTT reagent, as described above.All tumor cell lines were seeded in a 96-well plate for 24 h before performing the experiment at 37 °C, 5% COescribed . The celhttps://www.chemaxon.com accessed on 22 July 2022) and converted in 3D using the OpenBabel routines [The crystal structure of the human DNA-bound topoisomerase IB (PDB ID: 1T8I),  lacking routines . The recroutines . The proroutines . A molec−1∙Å−2 was imposed on the protein, DNA and ligand atoms, being slowly reduced, and finally removed in the last minimization cycle. The system’s temperature was gradually increased from 0 to 300 K in an NVT ensemble, using the Langevin thermostat [−1∙Å−2 was imposed on the protein, DNA and the ligand atoms, and then gradually decreased to relax the entire system. The systems were then simulated in an isobaric-isothermal (NPT) ensemble for 2.0 ns, using the Langevin barostat, imposing a pressure of 1.0 atm and maintaining the temperature to 300 K [The tleap module of the AmberTools19 program  was usedermostat , over a to 300 K . The SHAto 300 K . The pmeto 300 K . The cooto 300 K .GROMACS 2021.2 analysis tools  have beet-test. All data were expressed with mean ± SD values. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001.The cleavage and religation kinetics and dose dependent cell viability assay were analyzed by a two-way ANOVA test, using GraphPad Prism. The statistical analysis for the cell viability assay was evaluated by GraphPad Prism software, using the multiple To screen myricanol and its derivatives A–D againThe addition of DM to the hTop1 and DNA determines an inhibition of the relaxation activity, already detectable at a concentration of 80 μM A, lane 6The relaxation assay performed as a function of time with DM at a concentration of 100 μM, shows a lasting effect over time  in the absence and presence of DM, in a time course experiment, as in . BrieflyAnalysis of the religation step was carried out by incubating 0.6 pmol of the enzyme with 1.2 pmol of the suicide substrate to obtain the cleavage complex A, lane 2The results indicate that the presence of DM strongly inhibits the religation step of the enzyme catalytic cycle  simulation, to both validate the stability of the complexes and to correctly estimate the interaction energy between the hTop1-DNA and the drug. The results of the MM/GBSA calculations corroborated the strong interaction between DM and the hTop1-DNA structure, which is characterized by an interaction of free energy of −32.6 and −22.7 kcal/mol, for the drug inside the catalytic site or intercalated, respectively .Both vdW and electrostatic interactions contribute to the binding, although the electrostatic contribution is higher when the drug binds within the catalytic pentad. In order to provide an estimate of the contribution given by single residues or DNA bases to the total binding energy of the drug, the MM/GBSA per-residue decomposition analyses  were carVisual inspection of the trajectory indicates a partial rearrangement of DM within the binding site, which turns into a partially intercalated conformation , thus beDynamic cross-correlation maps (DCCM), based on the atomic fluctuations of 565 Cα atoms of hTop1, were generated from the three MD trajectories of the hTop1-DNA and hTop1-DNA-drug complexes, to evaluate whether the presence of the drug could induce changes in the structural-dynamical properties of the enzyme. In this representation, the positive values indicate a correlation of the motion between the two residues, which move in the same direction in space, while negative ones represent an anti-correlated motion, with residues moving in opposite directions. Indeed, MD simulations identify strong differences between the unbound and bound complexes and show the presence of functional structural motions in the hTop1 unbound structure . In the work of Tesauro et al., it was demonstrated that Ery-C is an in vitro catalytic inhibitor of hTop1, that efficiently acts on both the cleavage and the religation reactions . DM, a sf Ery-C, : this drThe inhibitory effect of the drug on different cancer cell lines, reported in Further experiments need to be performed to clarify the off-target mechanism, and additional chemical modifications need to be explored to improve the DM activity against hTop1, in order to develop a new anticancer drug targeting only hTop1, that could be used in clinic.


Targeted inhibition of aberrant signaling is an important treatment strategy in cancer, but responses are often short-lived. Multi-drug combinations have the potential to mitigate this, but to avoid toxicity such combinations must be selective and given at low dosages. Here, we present a pipeline to identify promising multi-drug combinations. We perturbed an isogenic PI3K mutant and wild-type cell line pair with a limited set of drugs and recorded their signaling state and cell viability. We then reconstructed their signaling networks and mapped the signaling response to changes in cell viability. The resulting models, which allowed us to predict the effect of unseen combinations, indicated that no combination selectively reduces the viability of the PI3K mutant cells. However, we were able to validate 25 of the 30 combinations that we predicted to be anti-selective. Our pipeline enables efficient prioritization of multi-drug combinations from the enormous search space of possible combinations.  •Systematics drug perturbations measurements in isogenic PI3K wild-type and mutant cells•Mutant-specific reconstruction and quantification of signal transduction networks•Short-term signaling response is predictive for longer term cell viability•25 out of 30 model predictions of mutant-specific multi-drug combinations validated Bioinformatics; Pharmacoinformatics; Systems biology; In silico biology The dependency of tumors on activated signaling pathways results in therapeutic responses to inhibitors that block pathway activity . Howeverin vitro testing of all combinations is infeasible. Computational approaches are required to prioritize promising combinations.These findings warrant further exploration of multi-drug combination strategies. This will require a systematic way to explore promising drug combination treatments, including optimizing the dosing of the different drugs. The combinatorial explosion of the search space — there are more than two million possible 4-way combinations of the 89 (as of 2020 ) FDA-appRecently, Nowak-Sliwinska and collaborators presented a “Feedback Systems Control” approach to explore the search space of possible multi-drug combinations . While tH1047R mutation knocked in under its endogenous promoter  and drug combinations were tested with both drugs at their IC50 values, to obtain a total of 34 different perturbations. We measured the response after 2 h of drug treatment (log2 fold change relative to DMSO control) of nine main nodes in the PI3K and MAPK signaling pathway using a multiplexed luminex assay to obtain more than 600 signaling drug-response measurements  (To establish how the PI3Kon (CNR)  of the MWe used the canonical MAPK and PI3K pathway interactions as prior information, and added four edges that were proposed by the CNR algorithm based on hyperparameters selected in a leave-one-out cross-validation loop A and S2AH1047R cells. These differences are highlighted in blue in H1047R (green) cells.  and phospho-ERK (pERK), which had a Pearson correlation with cell viability of 0.36 and 0.42, respectively A. The reWe therefore investigated whether a model combining the response of multiple nodes described the cell viability data better. To this end, we first fitted a linear model using all nodes as predictors We noticed that for both linear models there is a clear structure in the relation between the residuals and the fitted values A and S4B2 fold changes of pERK and pAKT upon drug treatment k. The parameters 2 fold changes of pERK and pAKT that cause 50% inhibition of cell viability. They differ slightly between the two cell lines, but the bootstrapped 95% confidence intervals strongly overlap  show no anti-selectivity, yet this combination is predicted to be one of the most anti-selective ones.  three-drug combinations are likely to have a mutant-specific impact based on a combination of single and two-drug high-dose drug-response measurements and mathematical modeling. We have used drug perturbation experiments to reconstruct, quantify and compare signal transduction networks of an isogenic cell line pair, and linked the responses of these networks to cell viability. No single signaling readout is highly predictive, but a non-linear model combining the response of pERK and pAKT gave a good fit. Importantly, this showed that the short-term signaling response is predictive for cell viability, which is measured in longer term experiments. Based on the so-obtained models, we were able to predict combinations that are specifically effective in one cell line but not another. 25 of the 30 of the combinations that we predicted to be anti-selective indeed were so in validation experiments, despite the fact that the differences between the cell lines in the training data were very subtle. It is to be expected that it will be easier to find mutation-specific drug combinations when the effect of a mutation on the signaling networks and on cell viability is stronger.50) individually show no selectivity toward the parental cells, the combination IGF1Ri + PI3Ki + GSK3i is one of the most anti-selective drug combinations, both as predicted by our model and as measured by validation experiments. This underscores the need for mathematical modeling in prioritizing promising combinations.All of the most strongly anti-selective drug combinations we identified contained the IGF1R inhibitor, but as monotherapy low-dose IGF1Ri is only mildly anti-selective. More generally, which multi-drug combinations are most selective or anti-selective is often far from obvious. For instance, while in the training data PI3Ki and GSK3i at their lower dose -proteins to hundreds of drug perturbations  in largeIn conclusion, here we have shown that it is feasible to make accurate, non-trivial predictions about (anti-)selectivity of multi-drug combinations based on mathematical models of signaling transduction networks. In combination with suitable model systems, this framework makes it possible to rationally design biomarker-selective low-dose multi-drug combinations.H1047R mutation more strongly than their parental counterparts, at least in this particular model system The absence of oncogene-specific sensitivities is presumably due to an absence of “oncogene addiction” .Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Evert Bosdriesz (This study did not generate new unique reagents.H1047R/+ MCF10A cell lines were obtained from Horizon discovery (HD PAR-003 and HD 101-011). Cells were cultured in DMEM/F-12 including 2.5 mM L-glutamine and 15 mM HEPES, supplemented with 5% horse serum, 10 μg/mL insulin, 0.5 μg/mL hydrocortisone and 0.1 μg/mL cholera toxin. Mycoplasma tests were performed every 2 months.Human parental and PI3KS133, EGFRY1068, ERK1T202/Y204, GSK3S21/S9, MEK1S217/S221, p70RSKT389, PRAS40T246 and RPS6S235 were purchased from ProtATonce Ltd. The luminex antibody against AKT1T473 was purchased from BioRad.The following inhibitors were used in this study: EGFRi (Gefitinib), IGF1Ri (OSI-906), RAFi (LY3009120), MEKi (Trametinib), ERKi (SCH772984), GSK3i (3F8), PI3Ki (BKM120), AKTi (MK-2206), mTORi (AZD8055). All inhibitors were purchased from MedKoo Biosciences. The luminex antibodies against CREB150 and IC90 values were fitted using the R-package MixedIC50 (https://github.com/NKI-CCB/MixedIC50).All the cell-viability measurements were performed in biological triplicates, each with 2 technical replicates, using black-walled 384-well plates (Greiner 781091). Cells were plated at the optimal seeding density (200 cells per well) and incubated for approximately 24 hours to allow attachment to the plate. Drugs were then added to the plates using the Tecan D300e digital dispenser. 10 μM phenylarsine oxide was used as positive control (0% cell viability) and DMSO was used as negative control (100% cell viability). Three days later, culture medium was removed and CellTiter-Blue (Promega G8081) was added to the plates. After 2 hours incubation, measurements were performed according to manufacturer’s instructions using the EnVision (PerkinElmer). Viabilities were normalized per cell line according to ixedIC50  . 300K cells per well were plated and incubated for approximately 24 hours to allow attachment to the plate. Drugs were then added to the plates and protein was harvested after 2 hours using the Bio-Plex Pro Cell Signaling Reagent Kit (BioRad 171304006M) according to the manufacturer’s instructions. Protein concentration of the samples was normalized after performing a Bicinchoninic Acid (BCA) assay , according to the manufacturer’s instructions. Cell lysates were analyzed using the Bio-Plex Protein Array system  according to the suppliers protocol as described previously . IntensiH1047R mutant cell lines were reconstructed based on the Luminex drug-response data using Comparative Network Reconstruction (CNR)(2 fold change of node i in response to perturbation k in cell line x) to the matrix of unobserved interaction strengths i with respect to node j in cell line x) and the direct perturbation effects k on node i in cell line x). These matrices are related throughMAPK and AKT signaling networks of the parental and PI3Kion (CNR). Brieflyion (CNR). It linkIn principle, ••Each inhibitor-target pair has a single indicator for the difference in perturbations strengths for both inhibitor concentrations, i.e. if •Most inhibitors are modelled as a perturbation to their direct target, i.e. EGFRi, MEKi, ERKi, GSK3i and AKTi are modelled as perturbations to EGFR, MEK1, ERK1, GSK3 and AKT1 respectively.•The MEK inhibitor interferes not only with MEK phosphorylation, but also with its catalytic efficiency. Hence, MEK inhibition was additionally modelled as a perturbation to its downstream proteins c.f. .•Some inhibitors target kinases that were not measured in our assay. The effect of these inhibitors was modelled as a perturbation to the  downstream nodes of the kinases being inhibited. Specifically, IGF1R inhibition was modelled as a perturbation to MEK1 and AKT1, PI3K inhibition as a perturbation to AKT1, RAF inhibition as a perturbation to MEK1, and mTOR inhibition as a perturbation to AKT1 and p70S6K.Additional constraints reflecting the experimental design were added to the CNR problem.Prior information about network topology was included by setting the indicators of a set of canonical MAPK and PI3K pathway interactions https://github.com/evertbosdriesz/cnr-selective-combos/blob/master/python/cnr-mcf10a-pi3k.ipynb.The full Comparative Network Analysis can be found in the Jupyter notebook under the following link: To obtain the distribution of residuals shown in Similarly, the distribution of residuals shown in The relation between signaling output and cell viabilityFinally, to obtain the distribution of residuals for random differences between the cell lines shown in k, 2-fold changes of pAKT and pERK relative to DMSO control upon perturbation k, respectively. The viability (relative to DMSO control) upon perturbation Equation vk=21+2−Rhttps://github.com/evertbosdriesz/cnr-selective-combos/blob/master/R/02-perturbations/mapping-signaling-drugresponse.Rmd.All code and details for this analysis can be found in the RMarkdown-file under the following link: k, i in response to this, CNR gives an estimate of the direct target inhibition of each drug only for the concentrations at which the drug was applied. To be able to simulate the effect of unseen drug concentrations, the relations between the applied concentration of drug The parameters  package . For con2 fold changes in response to a perturbation with drugs A, B and C, at concentration x relative to y was optimized by solving the following optimization problem:y should have under the 3-drug treatment, and that we (somewhat arbitrarily) set to 0.8. Specifically, to optimize selectivity we use For each possible 3-drug combination, the (anti)-selectivity for cell line Similarly, unselective control combinations were obtained by solving the optimization problem:A power analysis of the predictions was done by performing 1000 simulations with addition gaussian noise with a mean 0 and a standard deviation 0.25  to the results, and counting in what fraction there was an observable difference between the two groups.https://github.com/evertbosdriesz/cnr-selective-combos/blob/master/mathematica/optimize-combinations.nb.The optimizations were performed in Wolfram Mathematica  (version


Thymoquinone has been proved to be effective against neoplasms, including skin cancer. Its high lipophilicity, however, may limit its potential use as a drug. Melanoma remains the deadliest of all skin cancers worldwide, due to its high heterogeneity, depending on the stage of the disease. Our goal was to compare the anti-cancer activity of free thymoquinone and thymoquinone-loaded liposomes on two melanoma cell lines that originated from different stages of this cancer: skin-derived A375 and metastatic WM9. We evaluated the proapoptotic effects of free thymoquinone by flow cytometry and Western blot, and its mitotoxicity by means of JC-1 assay. Additionally, we compared the cytotoxicity of free thymoquinone and thymoquinone in liposomes by WST-1 assay. Our results revealed a higher antiproliferative effect of TQ in WM9 cells, whereas its higher proapoptotic activity was observed in the A375 cell line. Moreover, the thymoquinone-loaded liposome was proved to exert stronger cytotoxic effect on both cell lines studied than free thymoquinone. Differences in the response of melanoma cells derived from different stages of the disease to thymoquinone, as well as their different responses to free and carrier-delivered thymoquinone, are essential for the development of new anti-melanoma therapies. However, further research is required to fully understand them. Melanoma is a malignant neoplasm that arises from melanocytes, the cells that are responsible for the production of melanin, which absorb UV radiation . CutaneoThe achievements that have been made in the systematic treatment of melanoma provide the decrease of the death rates of patients in the USA in recent years. Nevertheless, melanoma is responsible for 0.6% of deaths caused by cancer worldwide, emphasizing the need for investigation of novel therapies . The choThymoquinone (TQ) is a member of monoterpenes, a group of chemical compounds containing one isoprenoid unit, and it is the main component of the essential oil of Nigella sativa (black seed), which for years has been known to display various beneficial properties. In the human body, it is metabolized to thymohydroquinone or to semiquinone, and the latter presents anti-cancer activities, increasing ROS in cancer cells ,9. AlthoIn recent years, even more studies highlight the possible effects of TQ in melanoma. The anti-cancer activity of TQ has been proved to be caused by various mechanisms, including a direct cytotoxic effect, enhancing apoptosis, DNA damage, and ROS formation. These effects have been attributed to the inhibition of Janus kinase 2 (JAK2) and the activator of transcription 3 (STAT3) signaling pathways ,16, as wDespite the advances in research into the effects of TQ on malignant melanoma, there have been few reports showing the effect of this monoterpenoid in human melanoma cells. Additionally, there have been no reports comparing its effects in cells derived from the neoplasms coming from different stages of the disease. Therefore, we evaluated the efficacy of TQ on two human melanoma models: cell culture derived from skin neoplasm and cell culture originated from melanoma nodular metastasis. Additionally, we compared the effectiveness of free TQ with the effectiveness of liposome-encapsulated TQ in the cell lines used. The presented study points out the differences and similarities in response to free TQ and TQ-loaded liposome treatment between the two melanoma cell lines.2, in a humidified atmosphere.A375  and WM9  human malignant melanoma cell lines were cultured in a high-glucose Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , 2 mM L-glutamine , 1 mM sodium pyruvate , and antibiotics . HEM   were grown in Melanocyte Growth Medium . All the cell lines were incubated at 37 °C, 5% CO4 cells/culture dish) were cultured for 24 h and then treated with free TQ (20 and 40 μM) for 48 h. Alternatively, the cells were pretreated with 10 μM Z-VAD-FMK general caspase inhibitor  for 1 h, before TQ treatment. Cells treated with DMSO were used as a negative control and those incubated with 1 μM staurosporine , as recommended for melanoma cells  × 100%. The readings were acquired from at least 4 independent experiments (each conducted in quadruplicate).The antiproliferative/cytotoxicity activity of free TQ and TQ in liposomes was evaluated using the Cell Proliferation Reagent WST-1 assay . In the present study, A375 and WM9 cells were seeded in 96-well plates .Data are presented as individual samples or mean ± standard deviation. Spearman’s rank correlation coefficient was used to evaluate the associations between TQ concentration and its effects. Differences between the TQ-treated and control groups, as well as differences between the two cell lines studied, were assessed by Mann–Whitney U test. A To assess the response of the different types of melanoma cells to free TQ, A375 and WM9 cell lines were treated with 20 and 40 μM TQ in DMSO for 48 h. The concentrations were chosen based on previously published reports , from whIn order to compare the sensitivity of both studied cell lines to TQ with their sensitivity to a known apoptosis stimulator, the cells were treated with 1 μM staurosporine, an inhibitor of protein kinases, for 48 h. A significantly higher percentage of apoptotic cells was shown for the population of the cells treated with 40 μM TQ than for staurosporine-treated cells, in the A375 cell line, but not in WM9 cells A,B.As far as the apoptotic stages are concerned, the late/early apoptosis ratio was significantly higher only in the A375 cell line treated with 40 μM TQ when compared with the DMSO control, whether or not the all-caspase inhibitor was used A. RegardTo compare the two tested melanoma cell lines in terms of the presence of apoptosis-associated proteins, we examined the expression of the cleaved PARP, an enzyme characteristic of the late stage of apoptosis, in the cells incubated with 5, 10, 20, and 40 μM TQ, by using Western blot A,C and sm) indicator. In healthy mitochondria, with normal ΔΨm, JC-1 is present as J-aggregates showing red fluorescence; whereas in unhealthy or apoptotic cells with low ΔΨm, the dye remains in monomeric form (J-monomers) that fluoresces in green. Consequently, the drop in the red/green fluorescence ratio indicates mitochondrial depolarization [m in both studied cell lines  for 48 h, or with 100 μM FCCP, an oxidative phosphorylation uncoupler, for 4 h (as recommended by the FCCP producer), as a positive control. Mitochondrial function was assessed by using JC-1 dye, a mitochondrial membrane potential . Both samples were characterized by homogeneous size distribution with a PDI of ca. 0.1. The determined zeta potential was −18.4 and −11.4 mV for non-loaded liposomes and TQ-LP, respectively. The negative charge of the liposomal membrane results from the presence of POPG, an anionic phospholipid. The determined values of the zeta potential point out the incipient instability of the system (Kumar and Dixit 2017); however, the measurements repeated 30 days after preparation did not show any particular changes in the liposome size or zeta potential during storage  by a thin lipid film hydration method. The parameters describing liposomal formulations obtained for the study are presented in  storage .The concentration of TQ in the TQ-LP measured by the gas chromatography/mass spectrometry method reached 1100 μg/L. The A375 and WM9 cells were treated with TQ-LP with the final concentration of TQ reaching 1.25 and 2.5 μM after 48 h. For a comparison, both cell lines were also incubated with free TQ (1.25 and 2.5 μM), as well as with a vehicle control (liposome) for the same period of time. A very strong negative relationship between the TQ-LP dose and cell viability was shown in both studied melanoma cell lines  with free TQ  for 24 h. For a comparison, A375 cells were treated analogously. TQ showed a selective antiproliferative effect on A375 cells, with a strong relationship between its dose and cell viability A. No staIn the last few years, there has been a renewed interest in compounds of plant origin as potential anti-cancer drugs, or as components of adjuvant therapy ,25,26,27To complete our knowledge of the effects of thymoquinone on human melanoma cells, we chose two different cell models for our experiments: an A375 cell line, originally isolated from the skin of a patient with malignant melanoma, and WM9 cells, derived from a metastatic site (an axillary node). Our results revealed that free TQ induced apoptosis in a dose-dependent manner, in both analyzed cell lines. Surprisingly, no difference in the percentage of apoptotic cells was observed in the two cell lines treated with TQ following the administration of an all-caspase inhibitor when compared to the cells that had not been pretreated with the Z-VAD-FMK. This, however, might be explained by a possible insufficient caspase blockage by the Z-VAD-FMK, which led to the execution of apoptotic cell death by the low level of active caspases . AlthougAs reported by Hatiboglu et al., TQ induced apoptosis by the blocking of the Janus kinase 2 (JAK2)/signal transducer and the activator of the transcription 3 (STAT3) pathway and, as a consequence, decreased the expression of anti-apoptotic proteins (Bcl-2 and survivin), and increased the expression of pro-apoptotic proteins, such as BAX and caspase 3, in B16F10 murine melanoma cells, within 24 h . Our stuAn important part of the middle apoptosis is mitochondrial outer membrane permeability (MOMP) that leads to membrane depolarization . In thisRegarding general cytotoxicity, TQ was proved to decrease cell viability in a dose-dependent manner in many cancer cell lines, including melanoma ,47,48. J50 value between 2.5 and 5 mg/mL, in A375 cells. The TQ-loaded liposomes used in our study proved to have much more cytotoxic properties than free TQ, with the lowest significant cytotoxicity observed at the concentration of 1.25 μM compared to 40 μM for free TQ. Notably, the cytotoxic effect of TQ-LP was shown for both A375 and WM9 cells, with no significant differences between the two cell lines. The results are consistent with previous reports on the use of TQ-loaded nanoparticles proving the lower toxicity of free thymoquinone in relation to the TQ packed in nanocarriers [The great pharmacokinetic advantage of TQ is its ability to penetrate the blood–brain barrier, due to a low molecular weight. However, to reach the adequate bioavailability of TQ, the usage of nanocarriers might be needed, as the molecule has poor water solubility and high lipophilicity ,51. Althcarriers . The lacTreatment recommendations for melanoma depend on many factors, including surgery, radiotherapy, and chemotherapy , and, usually, assume a systemic drug administration . RecentlIn conclusion, TQ proved to be toxic to both melanoma cell lines studied, with no apparent toxicity to normal melanocytes. Although the proapoptotic effect of free TQ was more evident in the A375 cells when compared to WM9, its antiproliferative activity proved to be stronger in the metastatic cell line. The incubation of A375 and WM9 cells with TQ-loaded liposomes resulted in much higher reduction in cell viability compared to free TQ, with no significant differences observed between the responses of the two cell lines. The knowledge of the differences in the response to free TQ and TQ-loaded nanocarriers of melanoma cells derived from different stages of the disease is crucial for the development of effective anti-cancer therapies. Further research using more in vitro melanoma models is needed to explore this topic.


Aspergillus, Penicillium and Fusarium. They can be synthesized on the field, during harvest as well as during storage. They are fairly stable compounds and difficult to remove. Among several hundreds of mycotoxins, according to the WHO, ochratoxin A, aflatoxins, zearalenone, deoxynivalenol, patulin, fumonisins as well as T-2 and HT-2 toxins deserve special attention. Cytotoxicity is one of the most important adverse properties of mycotoxins and is generally assessed via the MTT assay, the neutral red assay, the LDH assay, the CCK-8 assay and the ATP test in different cell lines. The apoptotic cell ratio is mainly assessed via flow cytometry. Aside from the assessment of the toxicity of individual mycotoxins, it is important to determine the cytotoxicity of mycotoxin combinations. Such combinations often exhibit stronger cytotoxicity than individual mycotoxins. The cytotoxicity of different mycotoxins often depends on the cell line used in the experiment and is frequently time- and dose-dependent. A major drawback of assessing mycotoxin cytotoxicity in cell lines is the lack of interaction typical for complex organisms .Mycotoxins are secondary metabolites of molds and mainly produced by species of the genera  Aspergillus, Fusarium and Penicillium. They can be detected in numerous foodstuffs, including nuts, spices, cereals and fruits, both pre- and post-harvest. Mold growth and, consequently, mycotoxin production can be affected by numerous factors such as weather conditions. The thermal stability and ability of mycotoxins to withstand food processing is one of the main reasons for concerns. Out of several hundreds of mycotoxins most commonly occurring and threatening human and animal health, we highlight patulin, aflatoxin, ochratoxin A, fumonisin, deoxynivalenol, T-2 and HT-2 toxins, zearalenone, citrinin and enniatin [Mycotoxins are toxic compounds mainly produced by species of the genera enniatin .Aspergillus and Penicillium, mainly by Penicillium expansum in rotten fruits and fruit juices. The temperatures at which P. expansum can produce patulin range from 0 to 24 °C [Patulin is a mycotoxin produced by several species of the genera Aspergillus species, mainly by Aspergillus parasiticus and Aspergillus flavus. They can be divided into Aflatoxin B1, B2, G1 and G2. These mycotoxins can be produced during storage as well as on the field, typically in warmer and more humid climates. Intoxication by aflatoxins can lead to immune system suppression, child development impairment, cancer or, in severe cases, death [Aflatoxins are produced by s, death .Penicillium verrucosum, Aspergillus ochraceus and Aspergillus carbonarius [Ochratoxin is a mycotoxin that occurs during storage under inadequate conditions. This mycotoxin is mainly produced by bonarius .Fusarium species, including Fusarium proliferatum and Fusarium verticilioides. Among several fumonisins, fumonisin B1 is the most common one [Fumonisins most commonly occur in dry and warm regions and often contaminate maize. They are mainly produced by mmon one .Fusarium culmorum, Fusarium graminearum and Fusarium nivale. It is generated during growth and harvest periods in relatively humid and cool weather conditions [This mycotoxin is one of the trichothecenes, produced mainly by nditions .Fusarium species. High levels of these toxins are found mostly in developing countries as they require high temperatures and relatively high humidity  to produce mycotoxins [The T-2 toxin and its derivative HT-2 toxin are further examples of trichothecenes produced mainly by cotoxins .Fusarium moniliforme, F. culmorum, F. graminearum, F. oxysporum, F. sporotrichides and F. crookwellence [Zearalenone is largely produced during the growth period in moderate climates with relatively high humidity. The main producers are wellence ,9.Penicillium, Aspergillus and Monascus. It most frequently occurs in rice (where it is responsible for yellow rice disease in Japan), cereals, fruits and cheese [Citrinin is a mycotoxin produced by several species of d cheese .Fusarium and comprised of Enniatin A, Enniatin A1, Enniatin B and Enniatin B1. They can be found in nuts, spices, fruits, cocoa, coffee, several grains as well as their products. Enniatins are considered as emerging mycotoxins [Enniatins are secondary metabolites mainly produced by the genera cotoxins .Cytotoxicity is defined as the ability to harm living cells, causing, among others, protein synthesis disruption or weakening of the cell membrane, ultimately leading to cell death (both necrotic and apoptotic) ,13. CytoSince only living cells can reduce MTT to formazan, the amount of formazan is used to assess the number of living cells . The CCKThe neutral red uptake  assay relies on the ability of living cells to absorb neutral red dye. The dye penetrates cells via non-ionic diffusion and is accumulated in the lysosomes. After washing, the dye is extracted from cells using acidified ethanol and measured spectrophotometrically. This assay relies on the capability of cells to retain a pH gradient which allows the dye to penetrate the cell membrane and retain in the lysosomes. Since only living cells have this ability, the amount of measured dye is directly correlated with the number of living cells . The AlaVarious different cell lines are used for cytotoxicity assessment. The cell lines used in the reviewed literature are summarized in All reviewed articles are summarized in In the case of patulin, MTT was the most frequently used test, and HepG-2 was the most frequently used cell line. Results showed a decrease in cells’ viability, as well as ROS formation increase, cell cycle arrest and p53 gene expression. IC50 values for patulin range from µM to mM, depending on experiment conditions, time of incubation and cell line used x is combination index for n components at x% of cell proliferation inhibitionD)j is the dose of n mycotoxins that causes x% cell proliferation inhibition in the combination(xD)j is the dose of each n mycotoxin individually that causes x% cell proliferation inhibition, a method derived from the median effect principle ,99. The CI < 0.9, CI = 0.9–1.1, CI > 1.1 generally indicate synergistic, additive and antagonistic effects, respectively. Additionally, when the CI indicates a synergistic effect, dose reduction indices (DRI) can be calculated. DRI indicate a fold of reduction of dose of each component at a given effect level compared to a dose of each component individually and can be calculated from equation [DRI is dose reduction indicesmD is median effect doseequation :(2)CIxn=In the case of combinations of two mycotoxins, MTT was the most frequently used test, while HepG-2 was the most frequently used cell line. Results showed that cytotoxicity of binary combinations strongly depend on components . Moreover, a concentration of individual components seems to have an effect on mixture cytotoxicity . Moreover, a concentration of individual components seems to have effect on mixture cytotoxicity . This problem could be partially solved by using primary cell lines instead of continuous cell lines  or by using 3D printed models of tissues to emulate the natural environment better.


B‐cell progenitor fate determinant interferon regulatory factor 4 (IRF4) exerts key roles in the pathogenesis and progression of multiple myeloma (MM), a currently incurable plasma cell malignancy. Aberrant expression of IRF4 and the establishment of a positive auto‐regulatory loop with oncogene MYC, drives a MM specific gene‐expression program leading to the abnormal expansion of malignant immature plasma cells. Targeting the IRF4‐MYC oncogenic loop has the potential to provide a selective and effective therapy for MM. Here we evaluate the use of bromodomain inhibitors to target the IRF4‐MYC axis through combined inhibition of their known epigenetic regulators, BRD4 and CBP/EP300. Although all inhibitors induced cell death, we found no synergistic effect of targeting both of these regulators on the viability of MM cell‐lines. Importantly, for all inhibitors over a time period up to 72 h, we detected reduced IRF4 mRNA, but a limited decrease in IRF4 protein expression or mRNA levels of downstream target genes. This indicates that inhibitor‐induced loss of cell viability is not mediated through reduced IRF4 protein expression, as previously proposed. Further analysis revealed a long half‐life of IRF4 protein in MM cells. In support of our experimental observations, gene network modeling of MM suggests that bromodomain inhibition is exerted primarily through MYC and not IRF4. These findings suggest that despite the autofeedback positive regulatory loop between IRF4 and MYC, bromodomain inhibitors are not effective at targeting IRF4 in MM and that novel therapeutic strategies should focus on the direct inhibition or degradation of IRF4. Constitutive activation of MYC signaling is detected in more than 60% of patient‐derived cells and one of the most common somatic genomic aberrations in MM is rearrangement or translocation of MYC.MYC locus, causing their aberrant proliferation.MYC transcription.One way to target the IRF4‐MYC axis is through upstream epigenetic regulators. Bromodomain and extra‐terminal (BET) proteins inhibitors have emerged as potential therapeutic agents for the treatment of hematologic malignancies.CREB binding protein (CBP) and EP300 are bromodomain‐containing histone acetyltransferases.Given the positive auto regulation loop between MYC and IRF4 in MM, we hypothesized that combining the two classes of inhibitors with distinct transcriptional effects would have a synergistic impact on MM cells. To confirm this, we explored the effect of combinations of BET and CBP/EP300 inhibitors on the viability of a panel of MM cell‐lines. To assess whether the protein and mRNA levels for MYC, IRF4 and their downstream targets following drug exposure were consistent with those expected from the IRF4‐MYC auto‐regulatory loop model, we compared their experimentally measured with their simulated expression in a network model of MM molecular interactions. We found that within the time frames used there is no synergistic effect on the viability of MM cell‐lines. For all inhibitors we experimentally measured largely unaffected levels of IRF4 protein and downstream target protein mRNA levels. These results are consistent with the continued presence of IRF4 protein in MM cells due to its long half‐life. Our network modeling of MM therefore suggests that cell death induced by CBP/EP300 bromodomain inhibition is not exerted directly through IRF4 but indirectly through MYC.22.1® Cell Viability Assay. Each experiment was reproduced 3 times per cell line.Cell viability assay and statistical analysis were performed as described in the supplemental methods. In brief, cell viability after inhibitors treatment was assessed using CellTiter‐Blue2.2β‐actin . HRP‐conjugated secondary antibodies: anti‐rabbit  anti‐mouse .Detailed protocols for western blotting are available in the supplemental methods. Primary antibodies: IRF4 , MYC  and 2.3RNA extraction, cDNA synthesis, and quantitative real time PCR was performed as in the supplemental methods.2.4To measure protein half‐life, cells were treated with 10 μg/ml cycloheximide for up to 72 h followed by western blotting. Detailed protocols are available in the supplemental methods.2.5https://github.com/SiFTW/MMModel/). Regulated reactions were modeled as described previously.Computational models were constructed using Ordinary Differential Equations and solved using MATLAB 2020a and ode15 s. All code, equations and parameters used in modeling are available on Github  and JQ1+SGC‐CBP30 (0.28–0.67 μM). However, treatment with SGC‐CBP30 alone (IC50 1.58–5 μM) and ISOX‐DUAL (2.15–7.70 μM) showed reduced efficacy. The poor inhibitory activity of ISOX‐DUAL could be explained by its reduced affinity for BRD4 and CPB/EP300 (IC50 1.5 and 0.65 μM) when compared to JQ1 and SGC‐CBP30.50 comparable with that of JQ1 alone. Similar results were obtained when treating the cells for 72 h  and one acute leukemia (OCI‐AML3) cells lines were treated for 48 h with different concentrations of these compounds. As shown in Figure h Figure . Taken t3.250 value  and MYC (CDK4 and hTERT) downstream targets. We treated the cells with a concentration of drugs corresponding to their IC50 value for 4, 8, 24, 48 and 72 h  but consistent reduction of IRF4 protein levels in response to treatment with the JQ1+ SGC‐CBP30 combination we then asked whether bromodomain inhibitors work through repression of MYC, IRF4 or both. To do so, we used gene and protein network modeling to simulate the effect of a drug acting on the transcription of MYC, IRF4 or both Figure  using th4In this work we studied the effects on MM cell‐lines of two classes of bromodomain (BET and CBP/Ep300) inhibitors, with putatively distinct transcriptional effects, with the aim to disrupt the oncogenic feedback loop between MYC and IRF4. Specifically, we wanted to evaluate the possibility that the combination of these bromodomain inhibitors would have synergistic impact on the viability of MM cells and on the transcription and protein levels of IRF4 and MYC.Our data showed that while the two BET inhibitors JQ1 and OTX015 showed the most effective inhibition on cell viability, the CBP/Ep300 inhibitor SGC‐CBP/Ep300 and the dual BET‐CBP/Ep300 inhibitor ISOX‐DUAL caused the least effect. Since the combination JQ1+SGC‐CBP30 has a stronger inhibitory effect on cell viability compared to the dual inhibitor alone this suggests that the limited effect of ISOX‐DUAL is caused by its reduced affinity for BRD4 and CPB/EP300. Our data also indicate that combining JQ1 and SGC‐CBP30 does not lead to synergistic or antagonistic cytotoxic effects on MM cell‐lines. In line with previous studies,Our data shows that IRF4 is characterized by a long half‐life in a panel of MM cell‐lines. Previous studies have shown a variability in the half‐life's values for IRF proteins .A growing body of preclinical and clinical evidence suggests that bromodomain inhibition could be an important therapeutic approach in a number of hematologic malignancies.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.https://publons.com/publon/10.1002/hon.3016.The peer review history for this article is available at No ethical approvals were required for the studies conducted in this article.Supporting Information S1Click here for additional data file.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Figure S5Click here for additional data file.


Andrographis paniculata (Burm.f.) Wallich ex Nees that has anticancer properties in various cancer cell lines. The present study aimed to evaluate andrographolide’s anticancer effectiveness and potential molecular pathways using a DBTRG-05MG cell line. The antiproliferative activity of andrographolide was determined using the WST-1 assay, while scratch assay and clonogenic assay were used to evaluate andrographolide’s effectiveness against the cancer cell line by examining cell migration and colony formation. Flowcytometry was also used to examine the apoptosis and cell cycle arrest induced by andrographolide. The mRNA and protein expression level involved in the ERK1/2/c-Myc/p53 signaling pathway was then assessed using qRT-PCR and Western blot. The protein–protein interaction between c-Myc and p53 was determined by a reciprocal experiment of the co-immunoprecipitation (co-IP) using DBTRG-05MG total cell lysate. Andrographolide significantly reduced the viability of DBTRG-05MG cell lines in a concentration- and time-dependent manner. In addition, scratch and clonogenic assays confirmed the effectiveness of andrographolide in reducing cell migration and colony formation of DBTRG-05MG, respectively. Andrographolide also promoted cell cycle arrest in the G2/M phase, followed by apoptosis in the DBTRG-05MG cell line, by inducing ERK1/2, c-Myc, and p53 expression at the mRNA level. Western blot results demonstrated that c-Myc overexpression also increased the production of the anti-apoptotic protein p53. Our findings revealed that c-Myc and p53 positively interact in triggering the apoptotic signaling pathway. This study successfully discovered the involvement of ERK1/2/c-Myc/p53 in the suppression of the DBTRG-05MG cell line via cell cycle arrest followed by the apoptosis signaling pathway following andrographolide treatment.Human glioblastoma multiforme (GBM) is one of the most malignant brain tumors, with a high mortality rate worldwide. Conventional GBM treatment is now challenged by the presence of the blood–brain barrier (BBB), drug resistance, and post-treatment adverse effects. Hence, developing bioactive compounds isolated from plant species and identifying molecular pathways in facilitating effective treatment has become crucial in GBM. Based on pharmacodynamic studies, andrographolide has sparked the interest of cancer researchers, who believe it may alleviate difficulties in GBM therapy; however, it still requires further study. Andrographolide is a bicyclic diterpene lactone derived from  Andrographis paniculata and is widely used as a traditional medicine in Malaysia, Indonesia, China, and India to treat diabetes and malaria [20H30O5 and 350.4 g/mol [Glioblastoma multiforme (GBM) is the deadliest and most difficult to treat brain cancer disease. Patients diagnosed with GBM have a one-year survival rate because of the limitations of current treatments, which have unfavorable side effects on patients and make it difficult to remove the tumor from a sensitive area of the brain . In addi malaria . AndrogrMore interestingly, andrographolide is a potential drug candidate for the treatment of any diseases related to the central nervous system (CNS) because it has a neuroprotective effect in the CNS by preserving the neuronal structure and function . It is aAccording to the literature, andrographolide can be developed further as an anticancer drug to treat cancer cells through various mechanisms such as apoptosis, autophagy, cell adhesion control, proliferation, migration, invasion, anti-angiogenic activity, and other miscellaneous acts . FurtherThe C-myelocytomasis (c-Myc) oncogene is a transcriptionally encoded gene that regulates the expression of over 100 target genes involved in apoptosis, cell cycle arrest, cell growth, survival, and differentiation . C-Myc iMoreover, the development of a new drug that can directly target c-Myc still needs to be explored because many researchers have attempted to explain the molecular mechanism of a new drug involved in activating c-Myc in human cancer. After all, information and knowledge regarding the expression and possible interaction of c-Myc in cancer remain insufficient. This review aims to provide information and knowledge related to c-Myc in the development of GBM. This study also aimed to determine the interaction between andrographolide and c-Myc, as well as how andrographolide is involved in upstream and downstream c-Myc signaling pathways in the human brain cell line, as there is no evidence of this having been yet studied. Then, this study aimed to investigate whether c-Myc or c-Myc’s target genes can be targeted by andrographolide for cancer therapy.50 values were determined at 13.95 µM for 72 h, which demonstrated the greatest cytotoxic effects and were chosen as the main active dose and time in the subsequent experiments. The LC50 values for the normal cell line cannot be computed because, as shown in 50 values below 5.8 μM at 24 h of incubation (Human brain cancer cell lines (DBTRG-05MG) and normal brain cell lines (SVGp12) were exposed to nine different concentrations of andrographolide (0.781–200 µM) for 24 h, 48 h, and 72 h A,B. The cubation B. The effect of andrographolide on cell migration changes of the DBTRG-05MG cell line was assessed using the scratch assay. 50 values) and 27.9 µM  of andrographolide at 72 h was observed. To confirm the effect of andrographolide in reducing cell viability of DBTRG-05MG cell lines, the colony formation of cell lines treated with 13.95 µM (LC50 (8.60%), and 2LC50 (8.95%). This study reveals that andrographolide induced cytotoxic effects through G2/M phase arrest in a concentration-dependent manner.Cell cycle arrest was determined by staining the DNA of apoptotic DBTRG-05MG cells with propidium iodide after treatment with different andrographolide concentrations (13.95 μM and 27.9 μM) compared to the control cell. The apoptosis analysis was elucidated through the flow cytometry technique using double staining with annexin V-FITC and PI. The scattered diagram was obtained from flow cytometry analysis of cancer cell lines after exposure to different andrographolide concentrations (13.95 μM and 27.9 μM) compared with the control cell C. There 50: 13.95 μM) and high (2LC50: 27.21 μM) concentrations of andrographolide, the relative expression of c-Myc significantly increased from 1.12-fold to 5.73-fold. The relative expression of ERK1/2 and p53 were also increased from 1.72-fold to 2.87-fold and from 1.48-fold to 2.25-fold, respectively, compared to untreated DBTRG-05MG cells. This finding was further analyzed via Western blot to determine the protein expression level of c-Myc, ERK1/2, and p53. 50: 13.95 μM) and high (2LC50: 27.21 μM) concentrations of andrographolide were increased, respectively. The upregulation of c-Myc markedly increased the expression of p53. Furthermore, c-Myc was implied to interact with p53 constitutively, and this interaction can be evaluated through co-IP.The effect of andrographolide on the relative expression of ERK1/2, p53, and c-Myc in DBTRG-05MG cells was evaluated by Quantitative Real-Time Polymerase Chain Reaction analysis (qRT-PCR). In The study on the interaction or relation between c-Myc and p53 protein, co-IP, and Western blot analysis was performed accordingly. The reciprocal co-IP assay was performed with the total protein lysate from the DBTGRG-05MG after exposure to the andrographolide. The current anticancer drugs in conventional treatments are becoming resistant to cancer, particularly in GBM, which is one of the uncommon brain cancers produced from glial cells of the central nervous system (CNS). GBM is also the world’s leading cause of death due to the limitations of current conventional therapies such as surgery, radiation, and chemotherapy . This ha50 of 13.95 μM at 72 h. This finding can be supported by a previous study that tested andrographolide using various cancer cell lines with LC50 values ranging from 2.25 to 44.25 µM [50 value 13.95 μM, obtained from this study, was in the acceptable range reported by the previous study. This result also indicated that andrographolide showed more cytotoxicity toward cancer cell lines than a normal cell line. However, these studies remain controversial because no previous study was reported on andrographolide’s effectiveness in reducing the cell viability of the DBTRG-05MG cell line. This is because many studies used other types of cell lines to mimic the GBM diseases, such as Uppsala 87 Malignant Glioma (U-87 MG) [In this study, the andrographolide’s effectiveness in DBTRG-05MG cell lines was assessed by a different assay that covers cytotoxicity, proliferation, and the molecular mechanism of cell death. The cell cytotoxicity and proliferation of DBTRG-05MG were successfully determined by WST-1, scratch, and clonogenic assays. This present study found that andrographolide significantly suppressed the cell viability of the DBTRG-05MG cell line in a time- and concentration-dependent manner. The cytotoxicity of DBTRG-05MG cell lines towards andrographolides was higher compared to SVGp12 normal cell lines with an LC44.25 µM . Hence, U-87 MG) , GBM8401U-87 MG) , which sThen, this study was continued with the clonogenic assay, which showed that andrographolide was able to inhibit the cell proliferation of DBTRG-05MG in a concentration-dependent manner by reducing the colony formation of cell lines over time. A few studies were conducted to evaluate the effect of andrographolide on the colony formation of human glioblastoma cell lines that involved a different type of cell line, such as U-87 MG . The preThis study also revealed that andrographolide induced a cytotoxic effect on the DBTRG-05MG cell line through G2/M cell cycle phase arrest followed by apoptosis in a concentration-dependent manner. The percentage of cells arrested at the G2/M phase in DBTRG-05MG cells after treatment of andrographolide at 13.95 μM for 72 h increased significantly compared with the control. Then, the apoptotic cell increased in the DBTRG-05MG cell line after andrographolide of 13.95 μM treatment for 72 h. According to our findings, andrographolide successfully suppressed the cell proliferation of the DBTRG-05MG cell line and was arrested at the G2/M phase, followed by apoptosis. Then, to gain information on the molecular mechanism involved in the cell cycle arrest and cell apoptosis after the DBTRG-05MG cell line was treated with the andrographolide, the ERK1/2/c-Myc/p53 signaling pathway was investigated in this study. This study was successful in detecting the mRNA and protein expression levels of c-Myc, ERK1/2, and p53 genes via qRT-PCR and Western blot analysis. Our finding successfully found that the upstream and downstream regulator genes of the c-Myc signaling pathway involved ERK1/2 and p53. The mRNA and protein expression level of ERK1/2 increased accordingly after andrographolide treatment compared with control. This finding was consistent with the previous finding that reported andrographolide can interact and activates ERK1/2 . The preOur findings showed that the expression of ERK1/2 was upregulated in DBTRG-05MG along with the upregulation of the c-Myc and p53 after andrographolide treatment. However, another study reported that andrographolide treatment suppressed lung cancer cell growth by downregulating c-Myc expression . Our finOur findings were extended with a co-IP analysis to assess the interaction of c-Myc with p53 via this signaling pathway. After being treated with andrographolide, c-Myc and p53 were able to form a protein complex in DBTRG-05MG; both proteins were bound together to initiate an apoptotic signaling pathway. The positive interaction between c-Myc and p53 also was discovered in the nasopharyngeal carcinoma (NPC) cell line for apoptosis . Then, aBased on co-IP results, c-Myc displayed a single band A, whereaOverall, evidence suggests that andrographolide at low concentrations induced cell cycle arrest and apoptosis via the c-Myc signaling pathway. This finding suggested that ERK1/2 acts as an upstream regulator, enhancing the activation of the c-Myc signaling pathway by modulating the activity of apoptotic proteins such as p53. Andrographolide, temozolomide (anticancer drug), and crystal violet were supplied by Sigma–Aldrich (USA). Andrographolide and temozolomide were dissolved in DMSO and kept at −20°C until needed. RPMI 1640, DMEM, trypsin-EDTA, and FBS were purchased from Gibco (USA). Penicillin–streptomycin was purchased from Nacalai Tesque (USA). WST-1 -2-(4-iodophenyl)-3-(4-nitrophenyl)- 2H-tetrazolium inner salt) was purchased from Roche (Germany). The PI/RNase staining buffer and Annexin V-FITC Apoptosis Detection Kit were obtained from BD Pharmingen (USA) and Invitrogen (Austria).v/v) Fetal Bovine Serum  and 1% penicillin–streptomycin . The cells were maintained in standard cell culture conditions, a humidified incubator with 5% CO2 at 37 °C.Human glioblastoma cell lines DBTRG-05MG and normal glial cell lines SVGp12 were supplied by the American Type Culture Collection (ATCC), USA. Both cell lines were used in this study and were cultured in complete RPMI and DMEM supplemented with 10% , with the final concentration of DMSO adjusted to 0.2% for all concentrations of andrographolide. After incubation, the old medium was discarded, and the cell was washed using PBS. Then, 100 µL of the sterile medium of complete RPMI was supplemented with 10% (v/v) Fetal Bovine and 1% penicillin–streptomycin, and each well-received 10 µL of cell proliferation reagent WST-1 . The plates were incubated in 5% CO2 at 37 °C for 1 h, and the absorbance (Abs.) was measured using the ELISA reader (Multiscan Spectrum) at a wavelength of 450 nm with a reference reading of 630 nm. The optical density values from vehicle-control-treated cells with 0.2% DMSO were considered as 100% of cell viability. The experiments were triplicated to check the consistency. The LC50 values were computed using the GraphPad Prism 8.0.1 software by plotting a cell viability versus concentration graph. The percentage of cell viability was calculated using the formula below:The cytotoxic effect of andrographolide was assessed by WST-1 assay in DBTRG-05MG and SVGp12 cell lines. Both cell lines were seeded into 96-well plates at 6 × 105 cells/ml. Cell lines were cultured in 5% CO2 at 37 °C overnight to allow them to adhere to the plate. After cells reached confluency at 60–70%, the plate was scratched accordingly with 1 mL pipet tips, and the cell was washed once with PBS. Then, the cells were given treatment with an LC50 concentration of andrographolide (13.95 µM) and without andrographolide that was only treated with 0.2% of DMSO as vehicle control. The treated cells were incubated in 5% CO2 at 37°C for 24, 48, and 72 h. The following incubation cell was fixed and stained with 0.5% of crystal violet in 25% methanol for 1 h. The crystal violet was rinsed with tap water and dried on plates for a few days at room temperature. The plate was viewed and photographed on an inverted microscope at ×10 magnification. The wound area was evaluated using ImageJ software.The DBTRG-05MG were harvested and seeded in 6-well plates at 4 × 105 cell/mL of cell density for each well. Then, the cells were incubated in 5% CO2 at 37°C overnight to allow them to attach to the plate. The cells were treated with varying concentrations of andrographolide (13.95 µM and 27.21 µM) and without andrographolide that was only treated with 0.2% of DMSO as vehicle control. The treated cells were cultured in 5% CO2 at 37 °C for 72 h. After incubation, the old medium was removed and the cells were rinsed with PBS. The fixation solution (acetic acid/methanol 1:7 (v/v)) was added to the plate and incubated for 15 min at 25 °C (room temperature). Following the removal of the fixation solution, 1 mL of 0.5 percent crystal violet in 25% methanol was applied to the plates. The plates were placed for 1 h at 37 °C in a 5% CO2 incubator. The crystal violet was removed by immersing in tap water to rinse it off. The plates were allowed to air dry on a tablecloth at room temperature for a few days and were further observed, and images were recorded for the stained monolayer. The formation of the colony was counted and analyzed with ImageJ software. The plating efficiency (PE) and surviving fraction (SF) were calculated by using the formula below:The clonogenic assay was performed to evaluate the effectiveness of andrographolide toward DBTRG-05MG by observing colony formation after treatment. The DBTRG-05MG was harvested using trypsin-EDTA and seeded in 6-well plates with 4 × 104 cells/mL. Then, the cells were placed in a 5% CO2 incubator at 37 °C overnight to allow them to attach to the flask. The cells were treated with varying concentrations of andrographolide corresponding to LC50 (13.95 µM)/2LC50 (27.21 µM) and without andrographolide that was only treated with 0.2% of DMSO as vehicle control. The treated cells were cultured in a 5% CO2 incubator at 37 °C for 72 h. Cell cycle arrests were assessed by PI/RNase staining buffer. The treated cells were harvested, fixed, and permeabilized with 70% ice-cold ethanol overnight. Then, the fixed cells were centrifuged to remove ethanol and washed once with ice-cold PBS. The cell pellet was resuspended in 0.5 mL of PI/RNase staining buffer and incubated for 15 min at room temperature. The cell cycle distribution was analyzed using BD FACS CantoTM Flow Cytometer  using MoD fit software , while apoptosis assay was assessed using annexin V-FITC/PI double staining. Cell pellets were obtained after treatment were collected and re-suspended with 0.1 mL of 1× Annexin V Binding Buffer (AVBB), 5 µL of annexin V-FITC, and 5 µL of PI staining added in the tube. The cells were incubated in the dark at room temperature for 30 min. Flowjo software was used to analyze the stained cells using a BD FACS CantoTM Flow Cytometer .The DBTRG-05MG were cultured in a T25 flask at a concentration of 20 × 1050 (13.95 µM)/2LC50 (27.21 µM) and without andrographolide that was only treated with 0.2% of DMSO as vehicle control. According to the official guidelines, the AurumTM Total RNA Mini Kit  was used to isolate total RNA from DBTRG-05MG cells. The NanoDrop 2000/2000c spectrophotometer was used to measure the RNA concentration . Electrophoresis on a 1% agarose gel was used to evaluate the integrity of total RNA. For use in qRT-PCR later, total RNA was kept at 80 °C.Total RNA was extracted from the DBTRG-05MG cell line treated with varying concentrations of andrographolide correspondent to LCThe qRT-PCR was carried out using a Real-Time PCR machine, an iTaq Universal SYBR Green One-Step Kit on Qiagen Rotor-Gene Q-Pure detection . All reactions on each instrument were optimized to obtain the best amplification kinetics. The RNA template of each sample representing one biological triplicate was diluted to a working concentration of 30 ng/µL for the qRT-PCR analysis. The reaction setup and program thermal cycling protocol were followed according to the manufacturer’s protocol. A 5-point 10-fold serial dilution standard curve of RNA was used to generate a standard curve, while PCR efficiency (E) values ranging from 96.67 % to 109.04% were calculated over all primer sets with average correlations (R2) of 0.99. Primers used in this study were designed using NCBI/Primer-Blast and Primer3Plus as follows: GAPDH, FW 5’-ACATCGCTCAGACACCATG-3’ and RW 5’- TGTAGTTGAGGTCAATGAAGGG-3’; ERK1/2, FW 5’-CATTCAGCTAACGTTCTGCAC-3’ and RW 5’-GTGATCATGGTCTGGATCTGC-3’; c-Myc, FW 5’- TCCTCGGATTCTCTGCTCTC-3’ and RW 5’-TCTTCCTCATCTTCTTGTTCCTC-3’; p53, FW 5’- GACACGCTTCCCTGGATTG-3’ and RW 5’-GACGCTAGGATCTGACTGC-3’. The housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene for comparing the relative expression of cDNA using the 2-△△CT method.50 (13.95 µM)/2LC50 (27.21 µM), along with cells treated with 0.2% of DMSO as vehicle control. The total cell lysate was isolated from DBTRG-05MG cells using the RIPA lysis buffer  according to the manufacturer’s instructions. The cells, both treated and untreated, were collected and washed twice in ice-cold phosphate-buffered saline (PBS). After, 1 mL 1× RIPA lysis buffer containing protease inhibitor cocktail was added to the cell pellet with occasional vortex. The lysed suspension was incubated for 30 min on ice and the following centrifugation at 10,000× g for 10 min at 4 °C was performed. The cell supernatant was transferred into new tubes immediately for further use in Western blot analysis and immunoprecipitation assays.The total cell lysate was extracted from the DBTRG-05MG cell line treated with varying concentrations of andrographolide correspondent to LCg. Total cell lysates obtained from the previous method were pre-cleared with 50 µL protein G Sepharose beads and incubated at 4 °C for 1 h, followed by gentle mixing using a rotating mixer. The pre-cleared lysate was recovered by centrifugation at 1200× g for 5 min and precipitated with the protein of interest antibody (Ab). The 5 µL of c-Myc Ab was added into pre-cleared lysate and incubated overnight at 4°C with moderate mixing on a rotating mixer. The immune protein complex was recovered by centrifugation at 1200× g for 5 min at 4 °C, then gently resuspended with cold lysis solution 3 times. The immune protein complex was recovered by centrifugation at 1200× g for 5 min at 4 °C, and the protein concentration was quantified by Bradford assay using a Bio-Rad protein assay kit. The immune protein complex was probed by Western blot analysis.The protein G Sepharose beads  were activated by rinsing three times with 1000 µL of lysis buffer and centrifuging at 150× TM ECL Western Blotting Substrate (Thermo Fisher Scientific) and were developed in a dark room. The relative density of the interest protein band to the control protein (α-tubulin) was analyzed with ImageJ.The equal amount of denatured protein sample (30 µg) for each well was resolved on the 12% polyacrylamide gels and transferred to Bio Trace™ NT Nitrocellulose Transfer Membrane (PALL) by electroblotting. The membrane was blocked with 5% skim milk (Sun Lac) in 0.05% TBS-T for 1 h and stored at 4 °C. The membranes were then washed three times with TBST for five minutes each time before being incubated overnight at 4 °C with primary antibodies anti-p53 (1:500), anti-c-Myc (1:500), and anti-tubulin (1:500). After that, the membranes were probed with HRP-conjugated secondary antibodies for 2 h at 4 °C. The signal protein bands were detected using a Piercep ≤ 0.05 was accepted as statistically significant.All the experiments were performed in triplicate, and the results were expressed as mean ± standard error of the mean (SEM). Other data were analyzed in GraphPad Prism version 8.0.1 using a two-way analysis of variance (ANOVA), proceeded by the Student’s t-test and Tukey’s test of one-way analysis. The This study successfully demonstrated the role andrographolide in inducing a cytotoxic effect in DBTRG-05MG cell lines in a time- and concentration-dependent manner. Further, andrographolide was found to reduce colony formation and delayed cell migration into the wound area. This study also evidenced that andrographolide induced a cytotoxic effect via G2/M phase arrest followed by apoptosis via the ERK1/2/c-Myc/p53 signaling pathway in a concentration-dependent manner. To our knowledge, this is the first work to show the mechanisms of andrographolide cell death in the DBTRG-05MG cell line. Because of its efficacy in inhibiting the DBTRG-05MG cell line, andrographolide also has the potential to be one of the anticancer treatments for glioblastoma multiforme disease. However, further study needs to be performed to evaluate the cytotoxicity of andrographolide in vivo and in vitro. The function of other regulatory genes that may interact with the c-Myc signaling pathway must also be investigated intensively.