Purpose: To find underlying mutations causing highly-activated Wnt activity in mammary tumor cell lines associated with rounded morphology indicative of stemness/EMT.Methods: Stemness of high Wnt cell lines was confirmed using qPCR on selected genes and microRNA profiling, followed by whole-exome sequencing of 3 high Wnt canine mammary tumor cell lines and 5 low/absent Wnt cell lines. Candidate genes were identified and their involvement in Wnt activity investigated using siRNA silencing.Results: The high Wnt cell lines had morphological and gene expression characteristics reminiscent of stemness. All individual cell lines had about 4000 mutations in the exome in comparison to the reference canine genome. The three high basal Wnt cell lines had 167 unique exome mutations. Seven of these mutations resulted in a SIFT score <0.2 of proteins related to Wnt signaling. However, gene silencing did not change the Wnt pathway activation. Renewed analysis with respect to putative relations to Wnt signaling revealed that P-cadherin (CDH3) had three mutations in the coding region of the extracellular domain and was associated with high Wnt signaling. Silencing by siRNA not only in lowered Wnt activity, but also decreased levels of phosphorylated cSRC and sP-cad, and changed cell morphology towards spindle cell appearance.Conclusion: It is concluded that expression of mutated CDH3 is associated with activation of cSRC, stabilization of ß-catenin and a rounded morphology related to a stemness/EMT phenotype. A decreased Wnt activity can be found also by cSRC inhibition, but CDH3 silencing has an additional effect on morphology indicating reversal of EMT. Breast cancer is one of the most frequently occurring cancers in the world and, with an estimated 1.7 million new cases diagnosed worldwide each year, it remains a global health challenge. Although intensive screening and improved treatment options have increased the 5 year survival rate, still roughly 30% of woman with breast cancer eventually die of their disease due to metastases resistant to therapy . Not onlDuring recent years, spontaneous mammary cancer in dogs has shown great value for analysis and developing treatment methods for EMT involved signaling mechanisms . The incMetastases can be formed from epithelial cells that undergo epithelial-mesenchymal transition (EMT) resulting in cells with phenotypically stromal cell characteristics. During EMT cells detach from neighboring cells and the basal membrane, obtain a rounded morphology and acquire migratory capacity. This process can also occur in cancer cells at the invasive front of the tumors . Among oAs the question remained unanswered why our cell lines show this highly basal activated Wnt activity and a rounded morphology despite high E-cadherin expression, we decided to perform a whole exome sequencing analysis to find mutations unique for the high basal Wnt active canine mammary tumor (CMT) cell lines and to relate candidate genes to upregulated Wnt activity. These candidate genes were further investigated using mRNA silencing experiments. At the end we found that the candidate gene P-cadherin, with three extracellular domain missense mutations, was causing both Wnt activity and responsible for the morphology changes. Silencing of these P-cadherin mutations in EC1 showed that the Wnt activity is reduced and rounded cells got their spindle cell morphology back. The mutations may result in a possible enhanced sensitivity for proteolytic cleavage of the extracellular domain of P-cadherin and so stimulate Wnt signaling and morphological changes.ALDH1A1 and LGR5 were expressed at significantly higher in the rounded Wnt active cells. Expression of the epithelial marker CDH1 was higher, whereas the expression of mesenchymal markers INTα5, INTβ1 and VIM was significantly lower in the cells with rounded morphology. FOSL1 expression, involved in migration and invasion was lower. The most striking differences in the miR expression profile related to Wnt signaling and stemness were the low expression of miR-34a, -146b and -196a in the high active Wnt canine mammary tumor cell line CMT-U27, and the high expression of miR-200b, -205 and 200c compared to the control cell line CIPm . Most of the mutations were G>A or A>G  score ≤0.1. CAMK2A, CSNK2A2 and RAC1 had a splice donor (SD) or splice acceptor (SA) mutation. CSNK1D was selected although it was only mutated in CMT-U27 and CMT9 (Supplementary Table 1). CDH3 had more mutations in both the high and low active Wnt cell lines but also three specific mutations in the high active Wnt cell lines in the extracellular domain (EC). The mRNA expression levels of these selected target genes were measured. Differences in expression levels were found among the cell lines  relative to the same construct with inactivation mutations in the TCF binding site (FOPflash)   and 3D. CDH3 gene. Screening the exome sequencing data showed 3 mutations in CDH3 with high SIFT scores, meaning that the impact of these individual missense mutations on protein activity is predicted to be low. Nevertheless, the influence of these three single amino acid substitutions was further investigated. The Integrative Genomics Viewer (IGV) showed for all three missense mutations more than two reads  of the published cDNA was tested on mRNA level, protein expression, cell proliferation and Wnt activity and on CDH3 target genes in CMT-U27 cells. Silencing CDH3 had a significant knock down on mRNA of 50% at 48 h and 85% at 72 h normalized to the MOCK  resulted in decreased P-cadherin protein expression  Wnt pathway  2,5-diphenyltetrazolium bromide (MTT) assay . Briefly, 15,000 CMT-U27 cells were seeded in 96-well Primaria plates (Corning) in DMEM/F12 with 10% FBS. After 24 h of incubation cell adhesion and spreading, the cells were treated with a siRNA either or both with dasatinib , or with GI 254023X both dissolved in DMSO to a final stock concentration of 10 mM. Dasatinib was added in a concentration range from 1 μM to 50 μM diluted in medium to a final concentration of 0.2% DMSO and incubated for 24, 48 and 72 h . 5 μM GI 254023X was added and incubated for 48 h in 0.1% DMSO. The absorbance was measured at a wavelength of 595 nm by an spectrophotometer Anthos Multimode Detector . IC50 curves were plotted with Sigma-plot version 12.5.CMT-U27 cells were seeded at a density of 600,000 cells in 6-well Primaria plates (Corning) and after 24 h transient transfected with the siRNA’s. After 24, 48 and 72 h cells were washed with Hank’s balanced salt solution (Invitrogen) and lysed and scraped with RIPA buffer . Twenty t-test for two groups  or analyzed by one-way ANOVA for multiple groups. A value of P < 0.05 was considered statistically significant.Statistical analysis was conducted using IBM SPSS Statistics version 24.0.0.1 . All values were expressed as means ± SD. Statistical differences were evaluated for normal distribution, using a Student’s


This paper examines the complexation of anti-cancer small interfering RNAs (siRNAs) by cationic carbosilane dendrimers, and the interaction of the formed complexes with HeLa and HL-60 cancer cells. Stepwise formation of the complexes accompanied by the evolution of their properties has been observed through the increase of the charge ratio (dendrimer/siRNA). The complexes decrease the viability of both “easy-to-transfect” cells (HeLa) and “hard-to transfect” ones (HL-60), indicating a high potential of the cationic carbosilane dendrimers for siRNA delivery into tumor cells. Therapeutic nucleic acids hold great potential for anti-tumor therapy. However, the natural barriers of a cell form considerable obstacles against the efficient transport of nucleic acids (NA) into cytosol or target compartments ,2,3,4. NAmong many types of dendrimers described so far, cationic carbosilane dendritic systems gain considerable attention as potential NA-based drug transporters, due to their good biocompatibility, chemical stability, highly controlled size, inert inner branches and high charge density on the surface. To date, their utility has been demonstrated for the efficient delivery of model antisense oligonucleotide-targeted mRNA of the Renilla luciferase gene , pro-apoHere we report on a detailed physico-chemical study of the formation and stability of dendriplexes formed by cationic carbosilane dendrimers and pro-apoptotic siRNAs. Also, we evaluated the cell cytotoxicity of the dendriplexes towards cancer cell lines of different permeability, namely, the suspension human acute promyelocytic leukemia cell line (HL-60) and the adherent human cervical cancer cell line (HeLa).Cationic carbosilane dendrimers with peripheral quaternary ammonium groups were prepared and characterized according to the reported method . The molO-tert-butyldimethylsilyl-protected RNA phosphoramidites (5-(ethylthio)-1H-tetrazole as an activator; the coupling time was 5 min) and with automated procedures being optimized for the synthesizer. Cleavage from a solid support, and the removal of protecting groups of oligoribonucleotides were performed under the conditions described in 12 (BDEF32) and its fluorescein isothiocyanate (FITC)-labeled analogue G2O3[SNMe3I]11-FITC, for the delivery of the specific anti-HIV-1 siRNA Nef to interfere with HIV-infectivity in human primary astrocytes. The work indicates that this type of transporters is a promising alternative to achieving very high transfection levels in “hard-to-transfect” astrocytes without causing cytotoxicity. The biodistribution of the dendriplex formed by the siRNA and the dendrimer loaded with FITC was studied in vivo in BALB/c mice. The dendrimer efficiently transferred the siRNA to mouse brains and it crossed the bloodαbrain barrier, showing its great potential as a drug transporter for the therapy of neurological disorders. Motivated by these inspiring results we have chosen this family of cationic dendrimers for the binding and transport of pro-apoptotic siRNAs to “easy-to-transfect” (HeLa) and “hard-to transfect” (HL-60) tumor cell lines. The cationic carbosilane dendrimers of the second and the third generations GnO3[SNMe3I]m ; n = 3, m = 24 (BDEF33)) were prepared according to the convenient procedure described in [1H- and 13C-NMR spectroscopy and mass spectrometry.The study  demonstrribed in . In brieCationic carbosilane dendrimers can bind siRNAs, mainly by the means of electrostatic interactions. The properties of the dendriplexes formed depend on their composition, i.e., on the charge ratio of the components taken. To study the ability of the dendrimers BDEF32 and BDEF33 to bind siRNAs, and the properties of the formed dendriplexes, and to find a ratio that could be considered optimal for the further cell experiments, the profiles of siRNA binding by cationic dendrimers were obtained by the means of several physico-chemical methods. The use of independent indirect methods to study siRNA binding is required, since each of these methods provides information on only on an exact property of the dendriplexes  [The early stage of the siRNA binding to the dendrimers was studied by means of the ethidium bromide intercalation assay (EBIA). The method is based on the intercalation of the fluorescent dye EB into a double strand siRNA (a binding site of 2–4 base pairs). These interactions result in a significant increase of EB fluorescence intensity, and they cause a blue shift in EB fluorescence emission maximum. Strong electrostatic interactions between cationic dendrimers and siRNAs lead to a higher affinity of the dendrimers to siRNAs. The following structural distortions of siRNA causes the displacement of intercalated EB from the duplex and subsequent quenching of EB fluorescence. Thus, EBIA is a sensitive method for monitoring even subtle changes in double-stranded siRNA structure . We obseThe reversible nature of the binding was confirmed by recovery of EB fluorescence intensity upon the treatment of the saturated dendriplexes with heparin, a natural polyanion . When thFurther evolution of the double-stranded RNA structure upon the binding with dendrimers was studied by CD spectroscopy. The CD spectra of the naked siRNAs indicate the typical curve of an A-form helix geometry, with a strong positive band at ~260–265 nm, and a negative band at ~210 nm a,b. SincIn addition, the complexation was studied by the means of an agarose gel retardation assay. This method is based on the mobility of the charged macromolecules (or complexes of molecules) in an electric field through a porous lattice formed by agarose gel, with the retardation of the molecules depending on their charge and mass. Herein, it allowed us to observe the non-complexed siRNA in samples. The electrophoresis data show that the siRNA was fully complexed by cationic dendrimers, being in 2-fold charge excess, which corresponded to a 6.7-fold molar excess (BDEF32) or a 3.3-fold molar excess (BDEF33), respectively . The comIn the experiments, we observed the initial moment when stabile supramolecular associates were formed. The increase of the dendrimer/siRNA ratio led to the further evolution of the dendriplex structure, with the reorganization of the particles and the increase of their size and surface charge. The changes could be monitored by the means of electrophoretic light scattering. The technique was based on the scattering of the laser light by nanoscale objects moving in the electric field. The value of the potential on the border of the solvate shell  calculated from the scattering data gives the idea of the surface charge of nanoparticles. The profiles had a sigmoidal shape; negative charges of siRNAs were compensated at the 12-fold excess of the G2 dendrimer, and at the 4-fold excess of the G3 one. A further increase of the dendrimer concentration led to a charge saturation at the 20-fold (G2) or 10-fold (G3) molar excesses of dendrimers .The results obtained from the biophysical assays described above are summarized in the 50, CR50) corresponding to the charge or molar ratio of the half-binding; and (2) the saturation point  corresponding to the charge or molar ratio where the measured parameter is no longer changed Whereas the former point is convenient for observing dendritic effects, the latter represents the properties of dendriplexes at the end of the complexation.In the complexation profiles, two points are of special interest: (1) half-effect point . In contrast, the zeta potential values continue to evolve even after the saturation achieved, as shown by other assays  or 2.4 (for G3), a strong positive dendritic effect is observed; CRsat is 6 for both generations).Analyzing the data from the assays studying the dendriplexes’ formation, one could find an interesting regularity in the results obtained: the binding and saturation values that were obtained by CD spectroscopy, EBIA, and agarose gel electrophoresis were quite close one to another . Howobserved  analyzesIt should be noted that the stepwise formation of dendriplexes have never been observed upon the binding of NAs to dendrimer-based nanoparticles. The surface charge of dendriplex nanoparticles is stabilized at relatively low CR/MR values, corresponding to the full NA binding. However, similar behaviors has been observed for amphiphilic PAMAM dendrons ,39 and dsat/MRsat obtained from the zeta potential profiles for the preparation of dendriplexes is likely preferential for the biological experiments . The AFM images of the dendriplexes prepared at the CR value of 5, which is close to the saturation region, are given in the One can speculate whether the dendriplex composition  is optimal for biological experiments. Indeed, the initial intention is to keep the dendrimer content in samples as low as possible, to avoid unwanted side effects, for instance, increased cytotoxicity. However, the use of higher dendrimer excess likely leads to the decrease of the particle size ,41,42, wAs target cell lines, HeLa  and HL60 (human myeloid leukemia cells) were chosen to study the influence of the cells’ characteristics on the transfection efficiency. The lines under study represented two major types of the human cancer cell lines: adherent (HeLa) and suspension (HL60) lines. Due to the peculiarities of the cell membrane composition, HL60 cells are known as hard-to-transfect , whereasBoth types of dendrimers exhibited dose-response cytotoxicities towards HeLa and HL60 cells. The cytotoxicity profiles looked similar for both cell lines, thus suggesting that leukemia cells (HL60) are principally permeable by dendrimers a,c. As eComplexes of carbosilane dendrimers and siRNAs were shown to penetrate efficiently across the cell membrane . Herein,Upon treating target cells with anti-cancer dendriplexes, several consecutive processes take place: dendriplexes’ endocytosis, endosomal escape, siRNA release and functioning, apoptosis induction . A deficDendriplexes containing pro-apoptotic siRNAs (at 10-fold cation excess) have been shown to decrease the target cell viability, with the cytotoxic effect being the dose-response . The modIt is worth noting that, unlike the expected result, scramble siRNA appeared to exhibit some cytotoxic effect. Such an artifact is explained by the partial pairing of the scramble siRNA strands with mRNAs of the genes that are essential for normal cell functioning , resulting in an inhibition of their translation . These eTo use dendrimers as carriers for therapeutic nucleic acids, their compositions should be optimized to achieve better efficiency. Along with other factors, the dendrimer:NA ratio appears to have a considerable effect on the biological activities of the dendriplexes. Using cationic carbosilane dendrimers and anti-cancer siRNAs as a convenient model, we have summarized the data on the siRNA complexation obtained by several indirect methods, to find the optimal composition for dendriplexes for the siRNA delivery. Based on our findings, we have suggested a scheme of stepwise dendriplexes formation consisting the NA binding step, and further rearrangements of dendriplexes upon the increase of the dendrimer excess. The dendriplexes containing pro-apoptotic siRNAs Bcl-2, Bcl-xL, and Mcl-1 induced the cell death in both HeLa and HL-60 cell lines. This evidences that cationic carbosilane dendrimers are versatile agents to transfect both adherent and suspension cell lines, with the latter being known to be resistant to common synthetic vectors. The results obtained provide useful practical data for the design of dendrimer-based gene therapy tools.


For that purpose, 1H-NMR spectra of both cellular extracts and culture media were combined with multivariate data analysis, bringing to light no less than 20 discriminant metabolites able to separate the metastatic metabolomes. The supervised approach succeeded in classifying the metastatic cell lines depending on their glucose metabolism, more glycolysis-oriented in the BRAF proto-oncogene mutated cell lines compared to the others. Other adaptive metabolic features also contributed to the classification, such as the increased total choline content (tCho), UDP-GlcNAc detection, and various changes in the glucose-related metabolites tree, giving additional information about the metastatic metabolome status and direction. Finally, common metabolic features detected via 1H-NMR in the studied cancer cell lines are discussed, identifying the glycolytic pathway, Kennedy’s pathway, and the glutaminolysis as potential and common targets in metastasis, opening up new avenues to cure cancer.In this study, metastatic melanoma, breast, and prostate cancer cell lines were analyzed using a  Cancer is a genetic or epigenetic disease resulting in altered metabolic activities compared to normal cells, including biological hallmarks such as the Warburg effect ,2, the oMetastasis describes a phenomenon where cancer cells disseminate from a primary site to a secondary site, a critical state responsible for about 90% of deaths in cancer patients . DependiThe metabolic flexibility of metastasis complicates the handling and treatment cares, with cells adapting to their surrounding environment to support tumor growth. Therefore, identifying the preferred metabolic pathways in different types of metastatic cancer cells could open up new avenues in metastatic treatment based on metabolic interventions. Nowadays, metabonomics approaches have been successful in detaching from the one-size-fits-all treatment to adaptive treatment in the cancer field . The met1H-NMR) spectroscopy is a non-destructive technic, highly reproducible, giving an overview of the metabolome based on the simultaneous detection of a wide range of low molecular weight (LWM) metabolites. Altogether, these metabolites draw a signature that is different for each metastatic cell line. After the digitization of spectral data, individual signatures can be compared through multivariate analyses. The high demanding metabolic cell lines used in this study were selected to cover a wide range of metastatic cancer features: The MCF-7 breast cancer cell line harbors estrogen receptors whereas MDA-MB-231 was triple-negative; the bone-metastatic PC-3 cell line was TP53 mutated, whereas the LNCaP metastatic cell line was androgen-dependent; finally, the considered BRAFV600E-mutated metastatic melanoma were either sensitive  or resistant (D10) to the standard of care BRAF/MEK combined inhibitors. This motley population of cell lines, which are gender-dependent or not, differ from one another in terms of oncomutation(s), tissue’s origin and metastatic dissemination site. Thus, all of them were considered in the present study in order to first obtain the most comprehensive view of the metabolome of those breast, prostatic, and melanoma metastatic cell lines and, secondly, to establish a new classification of metastases according to their metabolome. Thus, through this study, we hope to identify common biological pathways shared by the metastatic cell lines, but also some discriminating specific features, which all together could be used as potential targets for future therapeutic strategies. Hence, the importance of the biological pathways identified will be validated by the use of specific inhibitors in a cell viability test.Proton NMR . The separation observed among the experimental groups was perfectly highlighted on the scores plot  generated from the cellular extracts of melanoma , prostate cancer , and breast cancer  were integrated, and the numerical values exported to an Excel table. Then, a signal normalization step was performed, dividing each 0.04 ppm length descriptor by the total area under the curve (AUC) signal of their corresponding cell line. The normalized data were next integrated into the SIMCA-P+ multivariate data analysis software in order to highlight possible metabolic differences between the different cancer cell lines. No outlier possibly arising either from experimental bias or from technical issues was detected by the principal component analysis PCA-X  initiallres plot A, where ngs plot B displayN-Acetyl-Glucosamine. Furthermore, the resonances arising from both PCho and GPC were successfully separated from each other. The signature displayed in Due to overlapping or low signal intensity, some biologically relevant information can be hidden and escape the multivariate analysis. In such cases, further information can be collected directly from the spectra using the MestRenova Peak Picking tool. Thus, two extra metabolites were detected and added to the metastatic signature thanks to additional investigations, being aspartate and UDP-1H-NMR metabolites intensities was generated . MSEA is a strong tool used for data interpretation of metabolites set with relative concentration variations, indicating which biological pathway was more stimulated from one cell line to another. Thus, the analysis below highlighted the potential biological pathways related to the 13 identified metabolites A. The deFor instance, metabolic pathways such as “glutamate metabolism”, “malate-aspartate shuttle”, and the “Warburg effect” were informative on the origin of energy resources, either from glycolysis or OXPHOS. Other information provided from “arginine and proline metabolism”, “glycine and serine metabolism”, and “amino sugar metabolism” were indicative of the various usages of glucose in cancer cells and that the glucose tree structure can be quite different depending on the cancer type.1H-NMR analysis, namely lactate, myo-inositol, glycine, alanine, and UDP-N-Acetylglucosamine. This analysis was possible because of the culture media composition, uniform between the cell lines (excepting LNCaP), and containing glucose as the main carbon source. The results highlighted two main profiles between the studied cancer cell lines. In three BRAF-mutated cell lines , lactate represented more than 65% of the glucose-related metabolites AUC and was associated with high myo-inositol content and lower amounts in glycine, alanine, and UDP-N-Acetylglucosamine. Considering the remaining cell lines, the glucose was less directed towards lactate synthesis, with lactate rates reduced from 65% to 45% and with an increase in glucose-related amino acids and UDP-N-Acetyl-Glucosamine. The PC-3 glucose-related metabolism was quite different from the others, with the lesser amount of lactate between all the cell lines and intensive production in myo-inositol. A ratio B was appBMR DT, PC3, and MCF-7 cell lines that harbored a higher level of aspartate and a lower level of lactate, resulting in a small Glycolytic/OXPHOS ratio. The ratios of the D10BMR, MDA-MB-231, 451-Lu, and M229 took the opposite direction and were increased significantly, with levels >100. The 451-Lu was clearly the most glycolytic cell line based on the ratio analysis, with a value above 700. This finding is in adequacy with the PLS model described in The Warburg effect and the malate-aspartate shuttle were two pathways identified by the MSEA analysis, respectively, with the following metabolites lactate and aspartate. Lactate, the end-product of glycolysis, indicated that ATP production was favored via the glycolytic pathway, whereas aspartate, a metabolite part of the malate-aspartate shuttle used for translocating electrons across the mitochondria membrane indicated ATP production through OXPHOS. Therefore, it would be advantageous to know how cancer cells used glucose for ATP synthesis. In this respect, the lactate-to-aspartate AUCs ratio was indicative. The calculated ratio shown in 1H-NMR spectra of the culture media, returning high R2 and Q2 values . Groups separation was highlighted on the scores plot  was also carried out on the res plot A, where ngs plot  and was 1H-NMR analysis. Viability results are merged in In order to verify whether the biochemical pathways identified by the metabonomics approach were indeed critical for cancer cell survival and growth, specific chemical inhibitors were tested on each cell line and cell viability measured, except for LNCaP, which were not appropriate cells for such test due to their poor adherence properties. Thus, three inhibitors named CB-839, Hemicholinium-3 (HC-3), and Oxamate were used to, respectively, impact the glutaminolysis, the choline metabolism, and the lactate production. The established mechanisms of the considered inhibitors are displayed in V600E melanoma [i/MEKi therapy redirects the ratio in the D10BMR cell lines from glycolytic-orientated to OXPHOS-orientated, meaning that BRAF inhibition may switch the ATP production metabolism. Furthermore, the consumption of extracellular glucose and its intracellular handling also reflected the two metabolic profiles, increased in BRAF-mutated cell lines thanks to glycolytic preferred activation.Based on the lactate and aspartate intracellular levels, two metastatic subclasses were identified in terms of ATP synthesis, either more glycolytic- or more OXPHOS-oriented. As expected, the BRAF-mutated cell lines presented the most glycolytic-orientated NMR signatures, with a higher amount in lactate, the end-product of glycolysis, in both intra- and extracellular compartments , and a lmelanoma , a transmelanoma . It is wmelanoma ,25. Thermelanoma . Interes1H-NMR approach revealed two metabolites related to this energetic shuttle, namely creatine and phosphocreatine. Three metastatic cell lines  displayed reduced intensities for those metabolites in their NMR-based metabolomes. This observation suggests the limited use of this metabolic shuttle in those cell lines. Therefore, this feature could be used to classify metastatic cells since some of them preferably rely on the creatine shuttle. In addition, such a metabolic singularity could be targeted by specific inhibitors of the creatine shuttle [Phosphocreatine (PCr) is synthesized from creatine (Cr) by creatine kinase (CK) , a shutt shuttle . For tho shuttle ,31,32. P shuttle ,34. Inte1H-NMR signature gave an overview of this differential glucose handling. BRAF-mutated cell lines showed a common glucose-related profile clearly separated from the others, orientated in both lactate and myo-inositol production, indicating a glucose use for ATP and phosphoinositides productions, respectively. As discussed, all metastatic cell lines produced detectable levels of lactate, indicating sustained anaerobic glycolysis. However, BRAF-mutated cell lines showed the greatest lactate levels as a potential response to MAPK overstimulation, HIF-1-α stabilization, and sustained glycolysis. In addition, BRAF-mutated metastases overproduced myo-inositol, a precursor used in cancer cells for the synthesis of phosphoinositides, messengers involved in the phosphoinositide signaling system. These lipids family are located in the inner membrane of the cells and are involved in pro-tumoral key protein regulation such as PTEN and AKT, resulting in cell survival, proliferation, and growth [BMR, LNCaP) production. Glycine is produced from glucose through serine metabolism, via the action of phosphoglycerate dehydrogenase (PHGDH) [BMR cell lines in this category, because BRAFi resistance has been incriminated in the metabolic switch of melanoma cells, with an increase in oxidative metabolism and glutamine dependency [N-Acetylglucosamine, a metabolite derived from glucose and glutamine, in LNCaP and D10BMR cell lines indicated a post-translational ability of the metastatic cells, to add a N-acetylglucosamine group on serine and threonine residues of key proteins, promoting migration and invasion [1H-NMR signature may give some clues on how glucose is preferentially handled in different metastases, for phosphoinositides synthesis in glycolytic-orientated cells, and amino acids synthesis in OXPHOS-orientated cells. In addition, the production and detection of UDP-N-Acetylglucosamine indicate an O-glycosylation of key proteins promoting metastasis invasion.Cancer cells are highly addicted to glucose uptake and metabolism, which can be consumed through different pathways depending on their own genome mutations. Here, some glucose-related metabolites detected in the metastatic d growth . Interes (PHGDH) . PHGDH w (PHGDH) . Conside (PHGDH) . It is npendency . Finallyinvasion . A receninvasion . As a reOur experimental approach also picked up some biochemical pathways shared by the considered metastatic cancer cells, such as those involving the glucose-related metabolites, glutamine, and glutamate, and choline-related metabolites. Although the relatively low sensitivity of 1H-NMR (0.1 mM) is criticizable, nevertheless its ability to pinpoint the main metabolic hallmarks of a specific cell type makes it a suitable technic for cancer investigation. In this study, the NMR results highlighted the Kennedy’s pathway, the glycolysis, and the glutaminolysis as three relevant pathways showing different relative intensities according to the genetic profiles of the metastatic cancer cells.Enhanced glycolysis enzymes have been strongly correlated as a cancer hallmark, underlying the high-dependency of cancers to glucose uptake and metabolism. Indeed, GLUT-1 and GLUT-3 transporters presenting higher expression and activity (10–12 fold higher) in cancer cells as compared to healthy tissues . The endGlutamine, one of the most blood circulating amino acids, is overused by cancer cells to produce antioxidants, lipids through the de novo lipogenesis process, as well as purines and pyrimidines . GlutamiThe choline metabolism was also incriminated in our study, with both detection of phosphocholine (PCho) and glycerophosphocholine (GPC). A correlation between the choline metabolism enzymes and the choline metabolism metabolites have already been described in cancer. Higher amounts of tCho metabolites were detected in both prostate and breast cancer tissues, with respect to healthy ones, and associated with an increase in key enzyme activity and/or expression . It appeIn this regard, a cell viability strategy was investigated to jeopardize metastatic cells by using specific inhibitors of critical cancer cell pathways. Interestingly, our findings suggest that blocking simultaneously both glutaminolysis and lactate production, using CB-839 and Oxamate, disrupts metastatic cell viability. Nowadays, these inhibitors are mainly used alone, or in combination with the standard of care in cancer treatments. However, a few promising studies investigated the synergistic combination of these inhibitors and provided encouraging results, such as the couple CB-839/3-BP that demonstrated positive synergistic effects in tuberous sclerosis mice .This study provides a novel metastatic metabolome classification, highlighting common metabolic shares and differences between prostate, breast, and melanoma metastatic cell lines. The overview of the metabolome enabled the selection of three targetable pathways, being the glycolysis, the glutaminolysis, and the Kennedy’s pathway. As it turns out, the viability test validated the combined glutaminolysis and glycolysis inhibition, opening up an interesting approach in the metastatic metabolome disruption.BMR cell lines, prostate cancer was represented by the PC-3 and LNCaP cell lines, and breast cancer by the MCF-7 and MDA-MB-231 cell lines. The melanoma cell lines used in this study  were kindly provided from the laboratory of Lipid Metabolism and Cancer (KUL-Belgium), and used in the following publication (https://www.nature.com/articles/s41467-018-04664-0). The PC-3 (ATCC® CRL-1435™), LNCaP (ATCC® CRL-1740™), MDA-MB-231 (ATCC® HTB-26™) and MCF-7 (ATCC® HTB-22™) cell lines were purchased from ATCC. All cells, except LNCaP, were grown in 75 cm2 flask using DMEM High-Glucose  supplemented with 4 mM of Glutamine (ThermoFisher), 10% of fetal bovine serum (Gibco Lot 42G8378K), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (ThermoFisher). LNCaP condition was slightly different with the use of RPMI 1640 (ATCC 30-2001) instead as a growth media. Concerning the D10BMR melanoma cell line, two conditions were included depending on the presence or absence of the Dabrafenib/Trametinib therapy (Selleckchem). Thus, the D10BMR DT condition corresponded to the cells that were grown in the previous described media with an addition of 0.4 μM Dabrafenib and 0.1 μM Trametinib dose, whereas D10BMR were grown with the therapy and then deprived of it during 72 h for metabolic changes observations. Melanoma cell lines were composed of M229, 451-Lu and D10n = 12 wells were performed for each condition, and absorbance was read at 570 nm. The following metabolic inhibitors were used in this study: CB-839  solubilized in DMSO, Hemicholinium-3 and Oxamate (Sigma-Aldrich) directly solubilized in the culture medium. For viability testing, cells were first seeded in a 96-well plate (4-6 k cells/well) and grown for 4 days in 10% FBS media. Then, the media were removed and replaced by fresh media containing the metabolic inhibitor(s) for 72 h. After incubation, cells were washed twice with PBS and fixed with glutaraldehyde 4%. The staining was then performed using a 1% crystal violet solution, and cell wall permeabilized using Triton. A number of n = 6 samples per group. Culture media were replaced by fresh media with 10% FBS to standardize the NMR analysis 24 h before collecting the samples. The 24 h culture media were removed from each flask and stored at −20 °C. The remaining cells were washed twice with 7 mL cold D-PBS (Sigma-Aldrich), quenched using 3 mL of cold methanol, and then collected using a scraper. Cells metabolism was de novo quenched by immersing the cell pellet into liquid nitrogen for a few seconds before storing at −80 °C. A methanol: water: Chloroform 1:0.9:1 (3 mL) extraction was carried out on the cell pellet to separate polar metabolites from macromolecules. The polar phase was dried using a SpeedVacuum and was stored at −80 °C before analysis. Every cell line was grown to confluency before sample collection, with 2HPO4/0.04M NaH2PO4, pH 7.4) prepared in a mixture of H2O/D2O . The samples were centrifuged at 13,000 g for 10 min. 50 μL of a 7 mM solution of 3-trimethylsilyl propionic-2,2,3,3-d4 acid (TSP) reference prepared in 100% deuterium oxide was added to 650 μL of each supernatant. Finally, 700 μL of this final mixture was transferred into 5 mm NMR tubes prior to analysis. For the culture media preparation, 500 μL of each sample were mixed with 250 μL of phosphate buffer. The samples were prepared as described above, with an addition of 14 mM TSP solution. Acquisition of the 1H-NMR spectra were processed on a Bruker 600-MHz Advance spectrometer for 1H, using the NOESYPRESAT-1D pulse sequence. The same acquisition method was applied to the samples of cellular extracts and culture media using 256 scans.The intracellular dried samples were resuspended in 700 μL phosphate buffer . Phases and baseline of each spectra were corrected using the automatic and manual software tools. The water peak region ranging from 4.20 to 5.50 ppm was excluded, and spectra intensities were calibrated to the TSP chemical shift arbitrary fixed at 100. Spectra were binned in small subregions of 0.04 ppm width called descriptors, which gave rise to 220 descriptors for each spectrum. Next, the area under the curve (AUC) was calculated for each descriptor in all spectra to obtain numerical data. Each descriptor value was divided by the total area of the spectrum for normalization. Data were then processed for multivariate data analysis using the software SIMCA-P+ 12.0 . Principal Component Analysis (PCA) followed by a Partial Least Square Discriminant Analysis (PLS-DA) were carried out on the dataset. Possible group separations were observed in the scores plot and descriptors responsible for such separation on the loadings plot. Only descriptors with a VIP >1 were considered, and their corresponding metabolites were identified. To evaluate the quality of the model, two parameters were considered, R2, which corresponds to the « goodness of fit parameter » and explains variation in the data, and Q2, which is the « goodness of prediction parameter » and represents the predictive power of the model. Validation was carried out for the PLS model reliability using two tests: A permutation test followed by a cross-validation analysis of variance test (CV-ANOVA). Discriminant descriptors were correlated to metabolites using the Chenomx NMR suite software (version 8.1.1) and the Human metabolome database (HMDB). Because of the descriptor size of 0.04 ppm that can contain several metabolites chemical shifts, and also metabolites that have a low intensity, the multivariate data analysis was not able to detect all of the discriminant metabolites. A semi-quantification comparison of the spectra in the MestRenova software was then processed to find these new metabolites that can escape from our first analysis. p-values. Discriminant identified metabolites were converted to a heatmap in order to better visualize the metabolic signature between the cell lines. The heatmap was generated using the Morpheus online software. To identify the most changed pathways between the cell lines, a metabolic enrichment was performed on the discriminant metabolites using the online available software Metaboanalyst 4.0. The Homo Sapiens Pathway Library was selected as a reference, and the pathway analysis was investigated based on the p-values from pathway enrichment analysis and pathway impact values.The Peak Peaking tool of the MestRenova software was able to detect and precisely calculate the area under the curve (AUC) of all the peaks from each spectrum and was used for the metabolic signature validation. For each discriminant metabolite detected in the previous step, the AUC of one isolated chemical shift, previously normalized by the total AUC of each spectrum, was calculated using the Peak Picking tool in order to obtain numerical data. These numerical data were then processed in the R statistical software, using two non-parametric simultaneous tests on the data. Basically, a Kruskal—Wallis test followed by a Dunn test was performed. The Holm method adjustment was used in this validation process and applied to


Chelidonium majus L. and Berberis thunbergii DC. IC10, IC50, and IC90 doses were established toward hematopoietic cancer cell lines using trypan blue staining. Alterations in the expression of 18 apoptosis-related genes in cells exposed to IC10, IC50, and IC90 were evaluated using real-time PCR. Sanguinarine and Chelidonium majus L. extract exhibit significant cytotoxicity against all studied cell lines. Lower cytotoxic activity was demonstrated for berberine. Berberis thunbergii DC. extract had no influence on cell viability. Berberine, sanguinarine, and Chelidonium majus L. extract altered the expression of apoptosis-related genes in all tested cell lines, indicating the induction of apoptosis. The presented study confirmed the substantial cytotoxicity and proapoptotic activity of sanguinarine, berberine, and Chelidonium majus L. extract toward the studied hematopoietic cell lines, which indicates the utility of these substances in anticancer therapy.Isoquinoline alkaloids belong to the toxic secondary metabolites occurring in plants of many families. The high biological activity makes these compounds promising agents for use in medicine, particularly as anticancer drugs. The aim of our study was to evaluate the cytotoxicity and proapoptotic activity of sanguinarine, berberine, and extracts of  Papaveraceae, Ranunculaceae, and Berberidaceae families. Berberine is a tertiary, protoberberine-derived alkaloid occurring in Berberidaceae, Papaveraceae, Menispermaceae, Ranunculaceae, and Rutaceae families. Due to their toxicity, sanguinarine and berberine play a defending role against viruses and fungi that are pathogenic towards plants [Sanguinarine and berberine are two of the most intensively investigated isoquinoline alkaloids in terms of their use in medicine. Sanguinarine is a benzophenantridine-type alkaloid occurring in s plants ,2.Sanguinarine exhibits various pharmacological activities, including antibacterial , anti-inThe most intensively investigated of these is the anticancer activity of sanguinarine and berberine. Many pieces of evidence indicate that sanguinarine inhibits cell cycle and induce apoptosis in various types of cancer cells ,20. SangNumerous studies have evidenced the anticancer activity of berberine ,30. BerbClinical applications of sanguinarine and berberine are limited by their toxic effects . In IndiDespite the toxicity and mutagenicity of sanguinarine and berberine, the compounds are still extensively studied due to the possibility of synthesis of derivatives with reduced toxicity. In optimized doses, these alkaloids could exhibit potential therapeutic effects with limited side effects. A particularly important direction of research for this aspect is to determine the effect of the compound and mechanisms of action towards different types of cancer cells. Therefore, in this study we performed an assessment of cytotoxic and proapoptotic activities of sanguinarine and berberine on selected hematopoietic cell lines derived from various types of leukemia, including HL-60, HL-60/MX1, HL-60/MX2 (acute promyelocytic leukemia), J45.01 (acute T cell leukemia), U266B1 (myeloma), CCRF/CEM, and CEM/C1 (acute lymphoblastic leukemia). HL-60/MX1 and HL-60/MX2 cell lines are the multidrug-resistant derivatives of the HL-60 cells, therefore these cell lines were used to evaluate the effects of studied samples on cells resistant to anticancer treatment.Chelidonium majus L., Macleaya cordata Willd., Lamprocapnos spectabilis (L.) Fukuhara, Fumaria officinalis L., Glaucium flavum Crantz, Corydalis cava (L.) Schweigg and Körte, Berberis thunbergii DC., Meconopsis cambrica (L.) Vig., and Mahonia aquifolium (Pursh) Nutt. [Chelidonium majus L. and the highest amount of berberine was found in Berberis thunbergii DC. [Previously a screening of alkaloid composition was performed for nine species: h) Nutt. . The higrgii DC. . TherefoChelidonium majus L. and Berberis thunbergii DC., respectively.Plant extracts are very complex mixtures of various compounds, which could exhibit antagonism or synergy of biological activity. The application of plant extracts in pharmacotherapy has a higher risk of side effects than pure compounds, the advantage of which is a more predictable therapeutic effect. In this study, we evaluated the cytotoxicity and proapoptotic activity of both pure alkaloids, sanguinarine, and berberine, as well as the extracts prepared from plants containing high amounts of these alkaloids, Chelidonium majus L. and Berberis thunbergii DC. toward HL-60, HL-60/MX1, HL-60/MX2, CCRF/CEM, CEM/C1, J45.01, and U266B1 cell lines was evaluated by determination of IC10, IC50, and IC90 doses in trypan blue staining test  was marked as IC50 with an asterisk (IC50*). The IC90 values of berberine were determined only for the CCRF/CEM cells at130 μM and J45.01 cells at200μM. For the remaining cell lines, the IC90 dose could not be determined due to insufficient cytotoxicity of berberine even at the maximum possible dose (250 μM) when the viability of the U266B1, CEM/C1, HL-60, and HL-60/MX1 cell lines did not fall below 90%. For this reason, the maximum dose to which the cells were exposed was marked as IC90*. The most potent cytotoxicity of berberine was found toward CCRF/CEM, J45.01, and HL-60/MX1 cells. The HL-60/MX2 line was the least sensitive to the compound .Chelidonium majus L. extract showed differentiated cytotoxic effect depending on the study cell line. The strongest cytotoxic effect of Chelidonium majus L. extract was exerted on the J45.01, CCRF/CEM, and CEM/C1 cells, and the cells of the HL-60 and HL-60/MX2 lines were the least sensitive to this extract   . StandarBAK1, BNIP3, and CASP9 was observed in U266B1 cells after exposure to sanguinarine, berberine, and Chelidonium majus L. extract. For IC10 doses, the highest gene expression modulatory activity was exhibited by sanguinarine, but in higher doses of tested samples, the most affecting expression of studied genes was berberine. A higher dose of berberine and a lower expression of BCL2, BIK, BNIP2, and CASP3 were observed  were upregulated in cells exposed to sanguinarine in IC10 dose, but in cells exposed to IC90 dose of this alkaloid these genes (excluding BAK1 and MCL1) were downregulated  was observed in CCRF/CEM cells compared to other cell lines. In turn, the highest expression of MCL1 and TP53 was demonstrated in CEM/C1 and HL-60 cells, respectively. The highest induction of genes associated with apoptosis by IC90 dose of sanguinarine was also observed in the CCRF/CEM cell line. In HL60/MX1 and HL60/MX2 cells, sanguinarine caused the lowest alterations in the expression of the studied genes. In these cell lines, the highest increase in expression of studied genes was observed after exposure to IC10 of berberine. Expression of examined genes in cells exposed to a higher concentration of berberine and extracts of Chelidonium majus L. and Berberis thunbergii DC. was differentiated across studied cell lines. Exposure to Berberis thunbergii DC. extract (145.5 µg/mL) caused upregulation of B2M and downregulation of BAD and BNIP2 in all studied cell lines . The PARAFAC univariate analysis explained only 28.3% of the variance, so bivariate analysis was performed, explaining 50.4% of the variance. The obtained results allowed the isolation of IC10 concentration of sanguinarine as an outlier  was similar to the IC50 dose of the sanguinarine solution (0.1–1.05 μM) [Chelidonium majus L. extract, but sanguinarine does not occur in the highest amount in this plant [Inhibitory concentrations received for 1.05 μM) . These fis plant ,54.Chelidonium majus L. extract, as previously evidenced in HL60 cells [Chelidonium majus L. [Our research confirmed the cytotoxic activity of 60 cells . The resmajus L. .Berberine also exhibited cytotoxic activity against all examined cell lines, however it was weaker than sanguinarine. IC10, IC50, and IC90 doses of berberine were significantly higher than determined for sanguinarine and was characterized by a high variation between individual cell lines. Due to low cytotoxicity, we were not able to determine the IC50 dose of berberine for HL-60/MX2 cells and IC90 doses for HL-60, HL-60/MX1, HL-60/MX2, CEM/C1, and U266B1 cell lines. Cytotoxic activity of berberine toward U266B2 cell line was previously evaluated by Hu and collaborators using Cell Counting Kit-8 . The autIn our study, the cytotoxic activity of berberine was evaluated for the first time in relation to J45.01, HL60/MX1, and HL60/MX2 cell lines.Berberis thunbergii DC. extract, which contained a relatively high amount of berberine [Berberis thunbergii DC. extract is insufficient to induce a cytotoxic effect, which is supported by relatively high values of IC10, IC50, and IC90 concentrations (25.15 μM–250.45 μM) determined for berberine.The demonstrated cytotoxicity of berberine toward tested cells suggests the potential activity of erberine . HoweverChelidonium majus L. and Berberis thunbergii DC. on the expression of 18 genes associated with apoptosis was tested in hematopoietic cell lines. The similarities between administered doses and the response of the cells after 24 h exposure to the tested samples were also investigated.In the next step of the study, the influence of sanguinarine, berberine, and extracts from BAK1 and BNIP3 was the most frequently observed. In CCRF/CEM cell line, all studied genes were upregulated with logRQ > 0.5 after exposure to sanguinarine. Berberine also exhibited an ability to raise expression of studied genes, but to a lower extent than sanguinarine. All studied genes were upregulated in HL-60/MX2 cells after IC10 berberine treatment. The expression of TP53 was differentiated across studied cell lines exposed to IC50 of berberine. In CEM/C1, CCRF/CEM, and HL-60/MX2 cells TP53 was upregulated, but in other cell lines the downregulation of this gene was demonstrated. This indicates diverse TP53-mediated responses of studied cells after treatment with berberine. Upregulation of BAK1 and BCL2L2 was the most often observed effect in studied cell lines after treatment with Chelidonium majus L. extract.Exposure to sanguinarine, especially in IC10 dose, caused the strongest upregulation of studied genes associated with apoptosis. Upregulation of TP53 gene expression. This is a beneficial effect because the promotion of neoplastic transformation cancer is associated with a reduced expression of the TP53. These findings support evidence that sanguinarine has a strong antiproliferative effect on cells with TP53 gene dysfunction [TP53 was observed.PARAFAC analysis shows that IC10 dose of sanguinarine was an outlier among other doses of tested samples. Exposure to sanguinarine at this dose induced interesting changes in gene expression, which seem to be more unambiguous in interpretation and more beneficial than those induced after exposure to higher doses of the compound. Sanguinarine in IC10 doses induced an increase in function . In the Chelidonium majus L. extract on the expression of genes related to the apoptosis process is very similar to the effect of sanguinarine in the IC10 dose. This suggests similarities in the mechanism of apoptosis induction between sanguinarine and Chelidonium majus L. extract. Upregulation of CASP8 and CASP3 observed in cells exposed to IC10 of sanguinarine and IC10, IC50, and IC90 of Chelidonium majus L. extract indicated induction of apoptosis in the extrinsic pathway. The intrinsic apoptosis is probably prevented due to the accumulation of sanguinarine close to the outer side of the inner mitochondrial membrane during its stimulation. Sanguinarine neutralizes the effects of stimulation of the mitochondrial membrane, inhibits the synthesis of ATP, and breaks the oxidative phosphorylation [CASP9 expression observed in cells exposed to Chelidonium majus L. extract suggests the intrinsic course of apoptosis, probably conjugated with the extrinsic apoptosis pathway.The presented results show that the effect of rylation . An incrChelidonium majus L. extract and low sanguinarine doses is the increase in the expressions of BAK1, BAD, and BNIP3. BAK1 gene encodes the Bak protein, which participates in the formation of mitochondrial transmembrane channels and mediates the release of proapoptotic factors from the intra-mitochondrial space, including cytochrome c [BAD gene, has the ability to form heterodimers with antiapoptotic Bcl-2 and Bcl-xL proteins, enhancing the proapoptotic activity of the BAX and Bak proteins [BCL2 family. This protein is responsible for the neutralization of anti-apoptotic proteins after the onset of the mitochondrial permeabilizing membrane [BCL2 family is regulated by sanguinarine and Chelidonium majus L. extract via the BNIP3 protein. Sanguinarine seems to be the main factor inducing apoptosis of cells exposed to Chelidonium majus L., despite a lower amount of this compound compared to the amounts of other alkaloids in this plant [Another effect that clearly indicates promotion of the process of apoptosis in cells exposed to both chrome c ,23. The proteins . The BNImembrane ,61. It cis plant ,54.BAK1, BAD, and BNIP3 was lower, suggesting the lower proapoptotic potential of sanguinarine in higher doses.It should be noted that in the cells exposed to the higher doses of sanguinarine, the expression of TP53 gene, whose expression was strongly elevated in the CCRF/CEM cells (for IC50 by 303% and for IC90 by 342%), but in other cell lines after exposure to the IC50 and IC90 doses of sanguinarine was reduced.Both the PARAFAC analysis and the clustering with the Euclidean distances indicated that CCFR/CEM cell line clearly differed from other cell lines after exposure to all doses of sanguinarine. In this cell line, all doses of sanguinarine increased the expression of almost all analyzed genes. This is particularly important in the case of the Chelidonium majus L. extract. In these cells, as well as in the HL-60/MX1 cells, the anti-apoptotic BCL2 expression was lowered. The remaining cell lines responded with an increase in BCL2 gene expression after exposure to all doses of Chelidonium majus L. extract. This result indicates that the sensitivity of HL60 and HL60/MX1 cells to the proapoptotic action of Chelidonium majus L. extract is mediated by the downregulation of BCL2.PARAFAC analysis and Euclidean clustering showed that the HL60 line is an outlier after exposure to all doses of Berberis thunbergii DC. extract for the transcription of 18 apoptosis-related genes in hematopoietic cancer cell lines. Berberine is a particularly interesting compound because it is characterized by a lack of toxicity in relation to normal cells, while the cytotoxicity towards cancer cells were reported [In this research, we evaluated the effect of berberine and reported . Despitereported ,32, the CASP3 expression was observed in the CEM/C1, CCRF/CEM, and HL-60/MX2 cells after exposure to all doses of berberine. This result clearly indicates that the induction of cell death of these lines was in the apoptosis way. Moreover, an increase in CASP9 expression, observed in the case of all cell lines exposed to berberine in the IC10, IC50, and IC90 doses, suggests the induction of programmed cell death with the intrinsic pathway.In the current study, a high increase in The multidrug resistant HL-60/MX2 cell line was the most sensitivity to sanguinarine and was characterized by higher upregulation of proapoptotic genes compared to HL-60 and HL-60/MX1 cells, which exhibit weaker multidrug resistance potential , Table 2Chelidonium majus L. extract , which may explain a lower increase in the expression of the same genes. It may also suggest the possibility of proapoptotic activity of other compounds present in the extract, whose identification requires further investigation. Our results confirmed that berberine and the Berberis thunbergii DC. extract have an activity promoting the apoptosis with minimal cytotoxicity in studied concentrations. Exposure to the The limitation of this study is assessment of cytotoxicity and gene expression in exposed cells independently from cell population growth parameters, like doubling time or maximum growth rate. Gene expression was evaluated after 24 h of exposure, however, mRNA levels may change in shorter time  and be a result of complex interaction with other RNAs. The influx and efflux time in relation to concentration of study samples could also affect the outcome. The influence of these variables on the sensitivity of the cells to cytotoxic activity of studied compounds should be elucidated in further investigations with optimized time points of the exposure.Chelidonium majus L. and Berberis thunbergii DC. Understanding the mechanisms that accompany the change in the expression of the studied genes is substantial when trying to determine the effects associated with the interaction of sanguinarine, berberine and the extracts studied for their anticancer activity. The obtained results significantly enrich the knowledge of the antineoplastic activity of substances and may accelerate their introduction into the clinical trial phase in the treatment of various types of leukemia.Observations presented in current study may have a significant impact on the understanding of the regulation of apoptosis at the transcriptome level after exposure to sanguinarine, berberine and extracts from The reference compounds sanguinarine and berberine were of analytical grade from Sigma-Aldrich Company . The purity of each compound was more than 98%, according to the manufacturer. 10 mM stock solution of sanguinarine in DMSO was prepared. For the cytotoxic study, a series of dilutions was prepared with concentrations 0.8, 0.6, 0.5, 0.4, 0.2, 0.1 and 0.01 mM. The final concentration of sanguinarine in cell suspensions were 1000-fold lower. Due to the limited solubility of berberine in DMSO (maximum 50 mM), berberine dilutions were prepared in 25 mM, 10 mM and 5 mM concentrations.Chelidonium majus L. extract and 29.1 mg/mL concentration of Berberis thunbergii DC. extract. Due to a harmful effect of DMSO to cells [Plant material selection and procedure of extraction were carried out as previously described . For cytto cells , to the 2 per well) in standard conditions  for 24 h using Galaxy B incubator .HL-60 (CCL-240), HL-60/MX1 (CRL-2258), HL-60/MX2 (CRL-2257), CCRF/CEM (CCL-119) and CEM/C1 (CRL-2265) cell lines were from ATCC collection and were purchased from LGC Standards, UK. J45.01 (ECACC 93031145-1VL) and U266B1 (ECACC 85051003-1VL) cell lines were from ECACC collection and were purchased from Sigma-Aldrich Co. . Before experiments, 1 mL of cell suspension, containing 5–9 × 105 cells, was cultured in sterile 12-wells plates . The supernatant was discarded, to the cells were added 1 mL PBS  and centrifugation was repeated. The supernatant was removed and the cells were resuspended in 50 µL of PBS. Subsequently, 10 µL of suspension was mixed with 10 µL of trypan blue solution  and incubated for 1 min in 37 °C. The total number of cells, as well as the percentages of normal and death cells, were counted on Counting Slides using Automated Cell Counter (Bio-Rad). The experiments were performed in triplicates on independent cell lines passages and the mean values were calculated. The curves presenting relationships between cell viability and the concentration of the studied samples were prepared in order to determine IC10, IC50 and IC90 doses for each of the studied cell lines.After 24 h exposure to IC10, IC50 and IC90 doses of studied samples, cells were centrifuged at 800 rpm for 10 min and the supernatant was discarded. Total RNA was isolated from the cells using Chomczyński and Sacchi method  and TRI The quality and quantity of isolated RNA was assessed by NanoDrop2000 UV-VIS spectrophotometer with NanoDrop2000 Operating Software . RNA samples with A260/A280 ratio ranged between 1.8 and 2.0 were intended to further investigations.Synthesis of cDNA was performed using High Capacity cDNA Reverse Transcription Kit , according to the manufacturer procedure. Briefly, the following reaction mixture was assembled on ice: 2 µL of 10X RT Buffer, 0.8 µL of 25X dNTP Mix (100 mM), 2 µL of 10X RT Random Primers, 1 µL of MultiScribe Reverse Transcriptase (50 U/µL), 0.5 µL of RNase Inhibitor (40 U/µL), 1 µL of isolated RNA and 1 µL of DEPC-treated nuclease-free water. The reaction mixture was incubated in thermocycler Mastercycler Personal  in the following conditions: 25 °C for 10 min, 37 °C for 120 min and 85 °C for 5 s. Obtained cDNA samples were stored in −20 °C. B2M, BAD, BAK1, BAX, BCL2, BCL2L1, BCL2L2, BID, BIK, BNIP1, BNIP2, BNIP3, CASP3, CASP8, CASP9, MCL1, PMAIP1, and TP53) using real-time PCR method. cDNA samples were amplified using a 7900HT Fast Real-Time PCR System . Reaction mixtures contained 1.25 µL of gene-specific TaqMan probe  described in −ΔΔCt) [Gene expression analysis was carried out for 18 genes related to apoptosis (n −ΔΔCt) ,65. The Berberis thunbergii DC. extract due to the lack of cytotoxicity in relation to the tested cell lines.Clustering analysis with Euclidean distance and parallel factor analysis (PARAFAC) were applied to reduce the large dimensionality of the data and to assess the similarities between the doses of the tested compounds, between the analyzed cell lines, and between changes in the expression of examined genes after exposure to the samples. The analysis was performed using R programming software. The analysis did not include data obtained after exposure to the


Tamoxifen treatment of estrogen receptor (ER)-positive breast cancer reduces mortality by 31%. However, over half of advanced ER-positive breast cancers are intrinsically resistant to tamoxifen and about 40% will acquire the resistance during the treatment.In order to explore mechanisms underlying endocrine therapy resistance in breast cancer and to identify new therapeutic opportunities, we created tamoxifen-resistant breast cancer cell lines that represent the luminal A or the luminal B. Gene expression patterns revealed by RNA-sequencing in seven tamoxifen-resistant variants were compared with their isogenic parental cells. We further examined those transcriptomic alterations in a publicly available patient cohort.We show that tamoxifen resistance cannot simply be explained by altered expression of individual genes, common mechanism across all resistant variants, or the appearance of new fusion genes. Instead, the resistant cell lines shared altered gene expression patterns associated with cell cycle, protein modification and metabolism, especially with the cholesterol pathway. In the tamoxifen-resistant T-47D cell variants we observed a striking increase of neutral lipids in lipid droplets as well as an accumulation of free cholesterol in the lysosomes. Tamoxifen-resistant cells were also less prone to lysosomal membrane permeabilization (LMP) and not vulnerable to compounds targeting the lipid metabolism. However, the cells were sensitive to disulfiram, LCS-1, and dasatinib.Altogether, our findings highlight a major role of LMP prevention in tamoxifen resistance, and suggest novel drug vulnerabilities associated with this phenotype.The online version of this article (10.1186/s12885-018-4757-z) contains supplementary material, which is available to authorized users. Approximately two thirds of breast cancers are estrogen receptor (ER) positive. As the receptor stimulates proliferation of mammary epithelial cells, it is also an important target in anti-hormonal cancer therapy. One of the most prescribed ER antagonists for first line therapy is tamoxifen that has helped millions of women since its discovery 50 years ago . HoweverIn addition to its intended anti-cancer effects, tamoxifen is known to have both direct and indirect effects on the cellular lipid metabolism. It has been shown to reduce blood cholesterol levels  and to bOn the other hand, reprogrammed metabolism is one hallmark of cancer cells  and has In the present study, we have delineated mechanisms underlying tamoxifen resistance by extending our drug screening and exome sequencing analyses on tamoxifen-resistant cell lines  by perfo2.The development, characterization and culturing of the tamoxifen-resistant and their isogenic parental cell lines has been published previously . In brieRNA isolation, library preparation, sequencing, and data-analysis were done as explained in Kumar A et al. . BrieflyRNA-sequencing data analysis was performed as described in detail in Kumar et al.  and inclp-value less than 0.001 (1E-3) were accepted as significantly altered. Log2 ratios of differentially expressed genes of the cell lines were visualized using heat maps. In the heat map analysis, genes and samples were ordered using unsupervised complete linkage clustering with Euclidean distance measure [Public patient transcriptome data  was down measure . PatientParental and resistant T-47D cells were plated on 6-well plates and grown in their default media +/− 1 μM 4-OH-tamoxifen for 72 h in triplicates. The cells were scraped into 2% NaCl and the subsequent lipid extraction was based on the Bligh and Dyer method . Cell lyParental and resistant T-47D cells were seeded on coverslips and grown in the default media +/− tamoxifen for 72 h. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature, and permeabilized with 0.3% Triton X-100 for 5 min, followed by 30 min blocking with 3% BSA-PBS at 37 °C. The primary and secondary antibodies were diluted in 1% BSA-PBS and consecutively incubated for 60 and 30 min at 37 °C. For detection of free cholesterol, cells were stained with 0.05% filipin (Sigma)  in 10% F2 to remove the detection of debris in the image background. Starting from the detected nuclei, the segmented regions were propagated to cover cell cytoplasm stained with ceruloplasmin. The cells that touched the image border were discarded. Finally, spot detection was used to segment galectin-3 stained spots. The maximum radius of the spots was set to 1 μm. We defined cells with more than 1 spot as galectin-3 positive to exclude false positive detection. Further, we calculated the percentage of galectin-3 positive cells and the average spots per cell.In order to measure the integrity of lysosomal membranes, we performed the detection of damaged lysosomes by galectin-1 and -3 translocation according to the previously published protocol . We firsThirty-three compounds that target lipid and cholesterol metabolism, or induce LMP, were selected for the DSRT by literature and vendor research  diluted 1:200 in PBS and Hoechst for nuclei detection. Twenty-five fields-of-view per well were acquired with the PE Opera Phenix HCS system (PerkinElmer) in a confocal mode with the 40× water immersion objective (NA 1.1). Exposure time and laser power were kept constant for each individual staining across different cell lines and conditions. Images were analyzed using a custom pipeline to measure LipidTOXGreen signal and image-based DSS based on cell counts. Images were preprocessed by applying flatfield correction using CIDRE method , and theer 2.2.0 . First, p < 0.05. All comparisons of the measurement of triglycerides, free cholesterol and cholesterol esters, LMP assay, and LipidToxGreen staining can be found in Additional file For all experiment that were at least done in triplicates the values were expressed as mean ± SD. One way ANOVA was performed on the mean of each measurement followed by Tukey test to enable multiple comparisons between groups. Statistically significance was accepted as SERPINA1, PLXDC2, NAV1, DOCK10 and LRP1) with altered expression were detected in the luminal A cell lines , also changed its expression in the patient samples . CP (Ceruloplasmin), a gene that was highly upregulated in the resistant T-47D cell lines (240–290 fold-increase) as well as MCF-7 Tam1 (26 fold-increase), was also found to be overexpressed in all the 3 metastatic patient samples ranging from 12-fold increase (patient 2) to 50–57 fold increase , which is known to regulate several lipid droplet proteins, was upregulated in resistant cells. In addition, the ATP Binding Cassette Subfamily A Member 1 (ABCA1), which functions as a cholesterol efflux pump was downregulated  with antibodies detecting the lysosomal-associated-membrane-proteins 1 and 2 (Lamp1 and Lamp2). Based on this analysis, we observed that most of the free cholesterol accumulated into lysosomes. We also discovered an increase in the amount and size of lysosomes as well as divergences in shape compared to their typical round form seen in the parental cell lines Fig.  and b. AAs lysosomal integrity, with cholesterol content of lysosomal membranes and cathepsin D among its regulators, plays an important role in the induction of cell death , we moniGuided by our RNA-sequencing results we selected 33 drugs, known to affect the genes or pathways involved in lysosomal alterations and lipid metabolism as well as some drugs identified in our previous screen . All the clones responded to AZD8055, a dual mTOR inhibitor, although T-47D Tam1 and Tam2 showed reduced sensitivity. Atorvastatin, which inhibits HMGCoA reductase, did not affect the CTG DSS levels Fig.  but was To see whether any of the compounds are able to revert the lipid phenotype prior to reducing the cell viability (ATP-measurement) or induction of cell death (cell count), we specifically monitored the changes in neutral lipids by quantifying the average LipidToxGreen intensity per well  - independent manner, which leads to an increase in its expression [SERPINA1, which is accompanied by a slight increase of ESR1 transcription  is another mechanism by which cells avoid lysosomal induced cell death . We specALDH2, another target of disulfiram, is upregulated in T-47D Tam1 but not in Tam2 , encoding for a rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids, in the tamoxifen-resistant T-47Ds  was observed in tamoxifen-resistant cells Figs.  and 4c. ynthesis . The comTaken together, our results highlight that tamoxifen resistant cell lines can potentially be used as a representative model for studies of tamoxifen-resistant patients. We propose that the breast cancer cells can acquire tamoxifen resistance by dysregulation of different cellular pathways, dependent on their individual molecular phenotypes. Here, we highlight the inhibition of lysosomal membrane permeabilization as one of the mechanisms to avoid cell death, whereas an increase in neutral lipids may enable the further survival of these cells. We further propose that drugs targeting cellular antioxidation machinery may be able to overcome tamoxifen resistance. However, investigating the relevance of the proposed mechanism of acquired resistance in patients remains a challenge. Given the vulnerability of tamoxifen resistant cells to approved drugs such as disulfiram and dasatinib, it would be interesting to investigate whether these compounds could also be effective in clinical trials in tamoxifen-resistant breast cancer patients.Additional file 1:Antibodies used in this study. (XLSX 10 kb)Additional file 2:Drugs used in this study. (XLSX 11 kb)Additional file 3:Statistical analysis of triglycerides, free cholesterol and cholesterol esters, LMP assay, and LipidToxGreen staining. (XLSX 38 kb)Additional file 4:RNA-sequencing statistics. (XLSX 10 kb)Additional file 5:Figure S1. Tamoxifen-resistant cell lines display distinct expression changes. Tamoxifen-resistant clones derived from same parental cells (A) and of the luminal A subtype (B) differ in their expression changes. Venn diagrams show overlap in numbers and percentage of genes that are differentially expressed. (PDF 363 kb)Additional file 6:Detected fusion genes of parental and tamoxifen-resistant cell lines. (XLSX 40 kb)Additional file 7:Batch corrected CPM counts of cell lines and patients. (TXT 28313 kb)Additional file 8:p < 0.001. (XLSX 22 kb)Enriched pathways with adjusted Additional file 9:Drug Sensitivity Scores  of CTG mAdditional file 10:Mean of the mean LipidToxGreen intensity values. (XLSX 59 kb)


Mycoplasmas spp. is among major contaminants of eukaryotic cell cultures. They cause a wide range of problemsassociated with cell culture in biology research centers or biotechnological companies. Mycoplasmas are also resistant toseveral antibiotics. Plasmocin™ has been used to treat cell lines but Plasmocin™-resistant strains have been reported.InvivoGen has developed a new anti-Mycoplasma agent called Plasmocure™ in order to eliminate resistant Mycoplasmacontamination. The aim of this study was the selection of the best antibiotics for treatment of mycoplasma in cell cultures.Mycoplasma species were evaluated by microbiological culture (as the gold standard method), indirect DNA fluorochromestaining, enzymatic (MycoAlert™), and universal or species-specific polymerase chain reaction (PCR) detection methods.In this study, animal and human cell lines available in National Cell Bank of Iran, were treated with Plasmocure™. Thetreatment efficacy and cytotoxicity of Plasmocure™ were compared with those of commonly used antibiotics such as BM-cyclin, Plasmocin™, MycoRAZOR™, sparfloxacin and enrofloxacin. In this experimental study, a total of 100 different mammalian cell lines contaminated with differentMycoplasma spp. A moderatetoxicity was observed during Mycoplasma treatment with plasmocure™; But, after elimination of Mycoplasma, cells werefully recovered. Mycoplasma infections were eliminated by Plasmocure™, BM-cyclin, Plasmocin™, MycoRAZOR™,sparfloxacin and enrofloxacin. However, the outcome of the treatment process  varied among different antibiotics. Plasmocure™ is comprised of two antibiotics that act through various mechanisms of action than those inPlasmocin™. Two-week treatment with Plasmocure™ was enough to completely eliminate Mycoplasma infections in mammalian cell cultures especially for precious or vulnerable cells.The highest number of cured cell lines was achieved by using Plasmocure™ which also had the lowestregrowth rate after a period of four months. As a conclusion; Plasmocure™ might be considered an effective antibiotic to treat Mycoplasma spp. contaminations cause a wide range of economical and biotechnical troubles in cell cultures in biological research laboratories as well as biotechnology companies  is used to treat cell linesinfected by Mycoplasmas and related cell wall-lessbacteria. Plasmocin™ can also be used as prophylaxisfor Mycoplasma and other bacterial contaminations. However, some Mycoplasmas have been reported to be resistant to Plasmocin™ . Plasmocure™is comprised of two antibiotics that act through mechanisms different from those of Plasmocin™ . Two-week treatment with Plasmocure™ is enough to completely eradicate Mycoplasmas .Methods for eliminating c cells , 8. Threlemented , 10. Thasmocin™ . To eradoplasmas . In the www.celljournal.org). All cell lines were analyzed by indirect DNA fluorochrome staining , mycoplasma enzymatic detection kit , universal or species-specific polymerase chain reaction (PCR) detection technique and microbiological culture as the reference method. During the experiments, the cells were incubated at 37°C in 88% humidified air containing 5% CO2 and cultured in medium including 10-20% fetal bovine serum    , 14. In Mycoplasma contamination by above-mentioned methods. For every harvested cell line, Mycoplasma contamination was evaluated after 3-5 days of culturein an antibiotic-free medium. In order to confirm theabsence of contamination with other microorganisms, cell lines were examined through the quality control of microbiological culture , Roswell Park Memorial Institute medium 1640 , F12 nutrient mixture , McCoy’s 5A medium , eagle’s minimum essential medium , Leibovitz’s L-15 medium , earle’s balanced salt solution , horse serum , Trypsin-EDTA , fischer’s medium , penicillin/streptomycin , non-essential amino acid , oxalate, pyruvate, and insulin , human insulin (Sis), bovine insulin , human endothelial cell growth factor , MEBM/MEGM   medium, fibroblast growth factor-basic from bovine pituitary , 200 mM L-glutamine , 100 mM sodium pyruvate , oxalate, sodium bicarbonate , 2-mercaptoethanol , hypoxanthine , thymidine , epidermal growth factor ,granulocyte macrophage colony-stimulating factor (GM-CSF) recombinant human protein . At the beginning, culture media, FBS, trypsin and phosphate-buffered saline  were analyzed and checked for  culture , 15. Celibiotics .Mycoplasma contamination. All the cured cultures were re-examined for regrowth of Mycoplasmas for 4 months following the treatment , Plasmocin™ and MycoRAZOR™ were chosen according to the manufacturer’s instructions. Furthermore, sparfloxacin and enrofloxacin working concentrations were determined according to previously published reports , 16-18. reatment .Mycoplasma colonies, every 3-4 days (The suspended cells 1 ml) were added to 10 ml of Pleuropneumonia-Like Organisms (PPLO) broth medium  supplemented with 10% horse serum , 1% yeast extract agar , L-arginine , D-glucose  and cultured at 37°C for 48-72 hours. In the next step, PPLO medium was vigorously stirred to observe monotonous turbidity. After centrifugation at 1500 rpm for 15 minutes, the precipitate (100 µl) was transferred to a solid PPLO agar  culture plate and incubated at 37°C for 4-6 weeks. Microscopy observation was used to investigate the formation of non-typical colonies or egg form of  ml were Mycoplasma bodies were detected as polymorphous particles with bluefluorescence. For indirect staining, the supernatants of cell cultures that were suspected to be contaminated with Mycoplasma, were added to the Mycoplasma-free Vero cell line   , 20. Mycoplasma detection kit was used according to the manufacturer’s instruction. Briefly, the ratio of the ATPs level in each sample before (Reading A) and after (Reading B) the addition of MycoAlert™ substrate, was considered an indicator for the presence of Mycoplasma contamination. The presenceof contamination was proved if the Reading B/Reading A ratio was greater than 1  was also determined using PCR-based method as described previously , indirect DNA DAPI staining, enzymatic MycoAlert™ and PCR detection  methods. Vero cell line (NCBI C101a) contaminated with several Mycoplasma species, andMycoplasma-free Vero cell line (NCBI C101b) distinct from different sources, were prepared and Mycoplasma-free NSO (NCBI C142) cell line were evaluated by above-mentioned methods and confirmed as positive and negative controls, respectively. Three different Mycoplasma strains including M.hyorhinis, M.arginini and M.fermentans were detectedand identifiedin the positive control cells (Vero cell line contaminated with Mycoplasma (NCBI C101a) by species-specific PCR primers) (The control cell lines of the study were assessed for primers) , 21.2) was used for comparisons of two-bytwo in six groups. In Chi-square tests, the difference among the antibiotics for the treatment of Mycoplasma-infected cell lines was analyzed and interpreted. Differences with a P<0.05 were considered statistically significant.Statistical analysis was performed using SPSS 24.0 software . Non-parametric Chi-square test (χMycoplasmacontamination. The type of mollicutes in each cell line determined by PCR-based method indicating that 65/100 (65%) of the infected cell cultures was contaminated by one Mycoplasma species. Moreover, 19/100 (19%) samples were contaminated with two species and 16/100 (16%) were contaminated with three different species (Table S1) . M.hyorhinis was detected in 46/100 (46%) of the studied samples, M.arginini in 40/100 (40%), M.fermentans in 32/100 (32%), M.orale in 12/100 (12%), A.laidlawii in 6/100 (6%), M.salivarium in 4/100 (4%), M.pirum in 3/100 (3%), and M.hominis, M.genitalium, U.urealyticum and M.pneumoniae in 2/100 (2%). In this study, 100 different human and animal cell lines were randomly selected and assessed for Mycoplasma treatment process are summarized in Table S1  and Figure 2. Mycoplasma infections were eliminated by Plasmocure™, BM-cyclin (Roche), Plasmocin™, MycoRAZOR™,sparfloxacin and enrofloxacin in 91, 70, 66, 55, 33 and 15% of the contaminated cell cultures, respectively. Furthermore, decontamination was confirmed by PCR, as no Mycoplasma was detected in cured cell cultures 14 days after the completion of the treatment period. Mycoplasma regrowth (reinfection or recurrent infection) was observed in 3, 12, 17, 42, 62 and 83% of the cured cell lines four months after treatment with Plasmocure™, Plasmocin™, BMcyclin (Roche), MycoRAZOR™, sparfloxacin and enrofloxacin, respectively. According to the obtained results, the highest level (22%) of cell cytotoxicity (culture death) was observed among Plasmocin-treated cell lines. While, BM-cyclin (Roche), Plasmocure™, sparfloxacin, MycoRAZOR™andenrofloxacin were cytotoxic to up to 13, 6, 5, 3 and 2% of the studied cell lines, respectively . Overall, results reported on antibiotic treatments of cell cultures by different studies are summarized in Table 3. Mycoplasma contamination remains one of the majorproblems in cell culture laboratories. Mycoplasmascan cause significant biological changes in culturedmammalian cells. In fact, consequences of Mycoplasmacontamination are unpredictable and may affect molecularand cellular properties of the infected cells  and Plasmocin™ were used successfully. In case of mild contaminations, particularly for vulnerable or precious cells such as myeloma, lymphoma, hybridoma or primary cultures, MycoRAZOR™ along with fluoroquinolones (sparfloxacin and enrofloxacin) can be used as alternative antibiotics. Plasmocin™ (comprised of a macrolide and a quinolone) acts on the protein machinery and DNA replication by interfering with ribosomal translation and replication fork, respectively. BM-cyclin (Roche) binds to the 30S and 50S ribosomal subunits and inhibits protein synthesis. According to the manufacturer’s information, the bacteriostatic components of BM-cyclin (Roche) are pleuromutilin and tetracycline whereas Plasmocin™ is composed of a macrolide and a quinolone , and vice versa. In case of cytotoxicity and cell death, especially in severe and intensive contaminations with multiple uinolone , 31, 32lication .Mycoplasma infections. Similarly, Molla Kazemiha et al. , ciprofloxacin and MRA , in 65, 66.25, 20 and 31.25% of the cell lines, respectively. In addition, cytotoxicity was reported in 0, 10, 17.5 and 25% of the cell lines treated with ciprofloxacin, MRA, BM-cyclin (Roche) and Plasmocin™, respectively. Nevertheless, recurrent Mycoplasmas infection was observed in 10 to 80% of thestudied cell lines after four months. In another study done by Molla Kazemiha et al. , MRA and ciprofloxacin, respectively. It is noteworthy that, the risk of cell culture loss was 0, 12.5 and 17.5% for ciprofloxacin, MRA and BM-cyclin (Roche), respectively. However, 82.5 (for ciprofloxacin), 62.5 (for MRA) and 12% (BM-cyclin) of the treated cell lines showed Mycoplasma regrowth , sparfloxacin (62%) or MycoRAZOR™ (42%). Plasmocure™, BM-cyclin (Roche) and Plasmocin™ were effective especially in eliminationof M.hyorhinis, M.arginini, M.fermentans and M.orale which were resistant to the other antibiotics used in thisstudy. Plasmocure™ showed the lowest frequency (3%) of regrowth in our experiments while regrowth was observed in 17 and 12% of cell lines treated with BM-cyclin (Roche) and Plasmocin™, respectively. Plasmocure™, BM-cyclin (Roche), Plasmocin™ and MycoRAZOR™ effectively eradicated mollicutes and cured 91, 70, 66 and 55% of the cell lines, respectively. However, sparfloxacin and enrofloxacin were considerably less efficient as they cured only 33 and 15% of the cell lines, respectively. In this study, we observed high frequency ofPlasmocin™ caused the highest rate of culture death (22%), although, it targets the prokaryotic DNA replication and protein synthesis machineries which are different from those of eukaryotic cells. In addition, MycoRAZOR™ showed lower cytotoxicity on the studied cell lines (culture death of 3%), which might reduce the risk of culture loss. Therefore, it may be recommended as the first-line alternative especially in case of expensive or hard-to-obtain cell lines , 10, 34.Mycoplasmas. Thus, we suggest using two or more antibiotics in alternating periods for a successful treatment or eradication of Mycoplasma contamination. For example, BM-cyclin (Roche) or MycoRAZOR™ (inhibitors of protein synthesis) can be used alternately along with ciprofoxacin, enrofloxacin or sparfloxacin (inhibitors of DNA gyrase activity and DNA replication) with specified intervals during the treatment period. Finally, we observed that the combination of two or more antibiotic with different mechanisms of action makes the interpretation of the results more complicated. Based on our experiments, this might increase the risk of culture death or antibiotic resistance of Mycoplasma agent in comparison with other antibiotics for elimination of Mycoplasma contamination in cultured cells. As a conclusion, we recommend Plasmocure™ as an effective antibiotic for the treatment of Mycoplasma infections in mammalian cell cultures especially for precious or vulnerable cells. These findings may also help researches at biotechnology laboratories for selection of appropriate antibiotics for treatment of Mycoplasma contamination in cell cultures. This report suggests Plasmocure™ as a reliable anti-


Among the four cell lines, SNU1196 showed the highest mRNA and protein levels of hENT1. Expression of hENT1 showed a linear correlation with the log value of the half-maximal inhibitory concentration of gemcitabine. During incubation with gemcitabine, pretreatment with hENT1-specific small interfering RNA (siRNA) resulted in higher cell viability than that in samples pretreated with control siRNA. In a clinical evaluation, the median progression-free survival was 24 and 11 weeks among patients with strong and weak intratumoral hENT1 immunohistochemical staining (P = 0.05), and the median overall survival was 52 and 26 weeks (P = 0.15), respectively. In conclusion, this study showed that increased hENT1 expression is associated with a stronger toxic effect of gemcitabine on BTC cell lines. The clinical outcomes in this study suggest that increased intratumoral hENT1 immunohistochemical staining is a possible biomarker predicting better therapeutic effects of gemcitabine on patients with advanced BTC. Further studies are needed to determine the precise role of hENT1 in BTC.Gemcitabine is a principal chemotherapeutic agent for biliary tract cancer (BTC). Expression of human equilibrative nucleoside transporter 1 (hENT1) is regarded as a potential predictive biomarker for a gemcitabine response in some cancers. This study was conducted to investigate the association between hENT1 expression and the effects of gemcitabine on BTC cell lines and on patients with advanced BTC receiving gemcitabine-based chemotherapy. A total of four BTC cell lines, HuCCT1, SNU-478, SNU-1079, and SNU-1196, were tested. mRNA and protein expression levels of hENT1 were measured by quantitative reverse-transcription polymerase chain reaction and western blotting, respectively. Cell viability after gemcitabine treatment was measured in a chemosensitivity assay. For clinical assessment, 40 patients with unresectable or recurrent BTC who were treated with gemcitabine (1000 mg/m Biliary tract cancer (BTC) is a relatively uncommon malignant tumor that originates in the biliary tract including the intra- and extrahepatic bile duct and gallbladder. This cancer has a poor prognosis because of the advanced stage at diagnosis in many cases or frequent recurrence even after curative resection , 2. ThusGemcitabine is an analog of cytidine and a commonly used chemotherapeutic agent for various cancers, including BTC. Just as other nucleoside analogs, it is a prodrug that requires cellular uptake and intracellular phosphorylation. Human equilibrative nucleoside transporter 1 (hENT1) and human concentrative nucleoside transporters 1 and 3 (hCNT1 and -3) are important for the transport of gemcitabine into the cell. Inside the cell, gemcitabine is metabolized to active gemcitabine diphosphate and triphosphate by deoxycytidine kinase , 5. DuriRecently, intratumoral hENT1 has been reported as a candidate predictive biomarker for gemcitabine therapy responses in various cancers –11. NeveThe present study was conducted to investigate the association between hENT1 expression and the effects of gemcitabine both on BTC cell lines and on patients with advanced BTC who follow a GP regimen.2. Gemcitabine was provided by Lilly Korea  for in vitro experiments. The company was not involved in anything related to the study.Four BTC cell lines were analyzed in this study. The HuCCT1 cell line (intrahepatic cholangiocarcinoma origin) was purchased from the RIKEN BioResource Center . The SNU-478 (ampulla of Vater adenocarcinoma origin), SNU-1079 (intrahepatic cholangiocarcinoma origin), and SNU-1196 (extrahepatic cholangiocarcinoma origin) cell lines were acquired from the Korean Cell Line Bank . All the cell lines were cultured in the RPMI 1640 medium  supplemented with 10% of fetal bovine serum (Gibco) and 1% of a penicillin/streptomycin solution (Gibco) and were maintained at 37°C and 5% atmospheric COTotal-RNA isolation, cDNA synthesis, and quantitative reverse-transcription polymerase chain reaction (RT-PCR)hENT1 and GAPDH were as follows: hENT1, 5′-CAGGCAAAGAGGAATCTGGA-3′ and 5′-GGCCCAACCAGTCAAAGATA-3′; GAPDH, 5′-TTCACCACCATGGAGAAGGC-3′ and 5′-GGCATGGACTGTGGTCATGA-3′. The primers were designed by means of published sequence data from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and a BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi/). The Power SYBR Green PCR Master Mix (Applied Biosystems) served as the reaction mixture, in a 20 μL volume in each reaction capillary. The mRNA expression of the gene under study was normalized to the corresponding expression of GAPDH.Total-RNA was isolated from the cells using the RNeasy Plus Mini Kit , and cDNA was synthesized with the High Capacity cDNA Reverse Transcription Kit . The primer sets for RT-PCR analysis of Cells were washed rapidly with ice-cold PBS and lysed in RIPA buffer  containing 1 mM of the protease inhibitor PMSF  and the Protease Inhibitor Cocktail . The cell extracts were centrifuged at 13,000 rpm for 20 min. The total-protein concentration in the supernatant was measured with the BCA reagent . For each sample, equal amounts of total protein were denatured and separated by sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane. The membranes were blocked for 1 h in 5% skimmed milk powder solution composed of Tris-buffered saline (TBS) containing 0.1% Tween (TBST). Membranes were incubated overnight at 4°C with primary antibodies against hENT1  and β-actin . After three washes with TBST, the membranes were incubated with the appropriate horseradish peroxidase–conjugated secondary antibodies for 2 h . Bound antibodies were detected by means of the ECL reagent  and an X-ray film .Cells were cultivated in culture plates to 60–70% confluence. After a wash with PBS, the cells were treated with the Lipofectamine RNAiMAX reagent  mixed with hENT1-targeting small interfering RNA  or nontargeting control siRNA  at a final amount of 10 nmol in Opti-MEM (Gibco). At 72 h after siRNA treatment, cells were harvested to examine silencing efficiency and the effect of the hENT1 knockdown.50) were calculated in the SigmaPlot software.These hENT1 knockdown cells and control cells were seeded at 3,000–5,000 per well in 96-well plates. After overnight incubation, the cells were treated with gemcitabine (0–100 μM) for 72 h. Cell viability was determined using the Cell Titer-Glo Luminescent Cell Viability Assay Kit . Luminescence was measured on a LMAXII 384 microplate reader . Half-maximal inhibitory concentrations (IC2 and cisplatin 25 mg/m2) [Between June 2012 and May 2014, 193 patients with BTC who received gemcitabine-based therapy were identified via electronic medical records at Seoul National University Bundang Hospital. Among them, 153 patients were excluded due to the following: gemcitabine as adjuvant therapy, single gemcitabine or combination with other agents as palliative therapy, a dose different from that in the current first-line regimen  as previously reported .Grading of hENT1 staining was performed by an experienced pancreaticobiliary pathologist (HK) as follows: 2+, membranous staining in more than 50% of tumor cells; 1+, membranous staining in 5–50% of tumor cells; 0, no hENT1 staining or staining in <5% of tumor cells. Grade 0 or 1+ was regarded as low hENT1 expression, and grade 2+ was considered high hENT1 expression according to another study .in vitro experimental results shown are representative of three or more independent experiments. Pearson’s χ2 test or Fisher’s exact test was conducted to determine the differences between categorical variables. Continuous variables were compared by the Mann–Whitney U test. Kaplan–Meier analysis was carried out to generate survival curves and calculate the median survival periods, which were compared by the log rank test. A two-sided P value less than 0.05 indicated statistical significance. All statistical analyses were performed in GraphPad Prism 7.0  and in R software v3.4.3 (The R Development Core Team).The hENT1 in each cell line was measured in comparison to that in HuCCT1 cells by quantitative RT-PCR  that was used in the ABC-2 trial [This study has several strengths. First, we conducted both cell line experiments and clinical analysis. Owing to the limited number of studies on BTC cell lines, our results are important because we demonstrated that the experimental and clinical results were consistent. Second, all the patients in this study received the same chemotherapy regimen Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


MCTP1 gene in all resistant cell lines. We observed overexpression of the S100A3 and HERC5 genes in TOP-resistant cell lines. Increased expression of the S100A3 gene was also observed in CIS-resistant A2780 sublines. Overexpression of the C4orf18 gene was observed in CIS- and TOP-resistant A2780 sublines. A short time of exposure to CIS led to increased expression of the ABCC2 gene in the W1 and A2780 cell lines and increased expression of the C4orf18 gene in the A2780 cell line. A short time of exposure to TOP led to increased expression of the S100A3 and HERC5 genes in both sensitive cell lines, increased expression of the C4orf18 gene in the A2780 cell line and downregulation of the MCTP1 gene in the W1 cell line. Our results suggest that changes in expression of the MCTP1, S100A3 and C4orf18 genes may be related to both CIS and TOP resistance. Increased expression of the HERC5 gene seems to be important only in TOP resistance.Low efficiency of chemotherapy in ovarian cancer results from the development of drug resistance. Cisplatin (CIS) and topotecan (TOP) are drugs used in chemotherapy of this cancer. We analyzed the development of CIS and TOP resistance in ovarian cancer cell lines. Incubation of drug sensitive cell lines (W1 and A2780) with cytostatic drugs was used to determine the primary response to CIS and TOP. Quantitative polymerase chain reaction (Q-PCR) was performed to measure the expression levels of the genes. We observed decreased expression of the  One of the main reasons for the low effectiveness of chemotherapy in neoplastic disease is the development of drug resistance. A very good model to study this phenomenon is epithelial ovarian cancer (EOC), the most lethal gynecological malignancy . Most paCIS is the most frequently used antitumor agent and is used in lung, testis, and ovarian cancer chemotherapies, among others. CIS inhibits DNA replication and RNA transcription by interacting with the nitrogen atoms of the DNA, preferentially with the N-7 atom of deoxyguanylic acid, which results in intrastrand and interstrand DNA cross-linking , decreasABCG2 gene  and TOP [A2780TR1 and A2780TR2 (A2780 topotecan resistant)] were generated by exposing A2780 cells to CIS or TOP at incrementally increasing concentrations. The human primary ovarian cancer cell line W1 was established using ovarian cancer tissue obtained from an untreated patient. The W1 sublines resistant to CIS [W1CR (W1 cisplatin resistant)] and TOP [W1TR (W1 topotecan resistant)] were obtained by exposing W1 cells to CIS or TOP at incrementally increasing concentrations. The final concentrations used for selecting the resistant cells were 1000 ng/mL of CIS and 24 ng/mL of TOP and were twofold higher than the plasma concentrations of CIS and TOP two hours after intravenous administration. The increase in resistance according to parental drug sensitive cell lines were as follows: 8.9-fold for W1CR vs. W1; 20.0-fold for W1TR vs. W1; 4.1-fold for A2780CR1 vs. A2780 and 3.3-fold for A2780CR2 vs. A2780; 59.6-fold for A2780TR1 vs. A2780 and 48.5-fold for A2780T2 vs. A2780 as described previously ,20.v/v) foetal bovine serum, 2 pM l-glutamine, penicillin (100 units/mL), streptomycin (100 units/mL) and amphotericin B (25 μg/mL)] at 37 °C in a 5% CO2 atmosphere.All the cell lines were maintained as monolayers in complete medium . The graphs were made using Sigma Plot 11.0 .Changes in For amplification, 12.5 μL of Maxima SYBR Green/ROX qPCR Master Mix (Fermentas), 1 μL of each primer  , 9.5 μL t-test.The statistical analysis was performed using Microsoft Excel software. The statistical significance of the differences was determined by applying Student’s MCTP1 gene in all resistant cell lines suggests a non-specific role for MCTP1 in drug resistance. Expression of the C4orf18 gene was characteristic for A2780 drug-resistant sublines. The significance of these genes in the resistance to cytotoxic agents was not described previously and should be further investigated. In contrast, increased expression of the S100A3 and HERC5 genes was described in different cancers. Our results suggest that these genes may also be associated with the development of drug resistance in cancer.In summary, our results show the expression of new genes in response to CIS and TOP treatment in ovarian cancer cell lines. Decreased expression of the