I. verum, G. glabra, R. Frangula, and L. usitatissimum present significant reduction in in vitro tumor cell proliferation, suggesting these extracts as possible chemotherapeutical adjuvants for different cancer treatments.Experimental studies have shown that a variety of chemopreventive plant components affect tumor initiation, promotion, and progression and the main difference, between botanical medicines and synthetic drugs, resides in the presence of complex metabolite mixtures shown by botanical medicine which in turn exert their action on different levels and via different mechanisms. In the present study, we performed an in vitro screening of ethanol extracts from commercial plants in order to investigate potential antitumor activity against human tumor cell lines. Experimental results obtained through a variety of methods and techniques indicated that extracts of The plant kingdom contains a great source of new bioactive compounds which, due to their intrinsic biological properties, may be used in medicine as well as in other human health promoting areas. Phytopharmaceuticals play an important role in general medical practice for the treatment of diseases of the cardiac and vascular system , nervousThe increase in interest in apoptosis, or programmed cell death (PCD), has had a major impact on many fields within the biological sciences, including oncology , 11. The Illicium verum, Glycyrrhiza glabra, Rhamnus Frangula, and Linum usitatissimum extracts induce downregulation of antiapoptosis genes at the same time that leads to a strong cytotoxic effect against the majority of tested tumor cells.In the present study, we report comparative data regarding the in vitro cytotoxic effect of different plant extracts on a panel of human tumor cell lines. Our results indicate that the (MCF-7), and the human colon carcinoma (HT29) cell lines were purchased from American Type Culture Collection . Dulbecco's modified Eagle's medium (DMEM), Leibovitz's L-15 (L-15), penicillin, streptomycin, gentamicin, and L-glutamine were from GIBCO BRL; HEPES was from United States Biochemical; fetal bovine serum was from HyClone; the apoptosis inducers, well characterized and effective, for example, in disrupting mitochondrial transmembrane potential and activating caspases as well as inducing phosphatidyl-serine (PS) exposure, DNA fragmentation, and other apoptotic characteristics, were from PromoKine.The human liposarcoma cell line (SW872), the human synovial sarcoma cell line (SW982), the human bone osteosarcoma cell line (HS 39.T), the human connective tissue leiomyosarcoma cell line (HS 5.T), the human acute promyelocytic leukemia cell line (HL-60), the human melanoma cell line (M14WM), the human breast adenocarcinoma cell line  were purchased from a local herbalist in La Coruña and belonged to the pharmaceutical company Soria Natural, Soria, Spain. 50 grams of seeds, roots, or leaves was mechanically powered and extracted with 70% ethanol for 48 h at room temperature in a percolator. After this incubation extracts were filtered and evaporated with rotary evaporator to render the extracts alcohol free. All the extracts were weighted, collected in amber vials, and kept in refrigerator until being used.All plants used in the study2 humidified atmosphere and 95% air in a CO2 incubator. Cells were passed twice/week under sterile conditions.All cell lines were grown as a monolayer culture (except HL-60 which grows in suspension in RPMI 1640 medium) in DMEM , L15 , and MEM (M14WM) supplemented with 1% nonessential amino acids, 1% L-glutamine, 100 IU per mL penicillin, 100 IU per mL streptomycin, 20 mg/mL glutamine, and 10% fetal bovine serum (20% for HL-60 cell line) at 37°C in a 5% CO5/mL in culture medium (with 10% FBS) was used for the experiments in 96-well microtitre sterile plate. To each well 100 μL of cell suspension was placed. The test extract was added at different concentrations  and the viable count was done by Trypan blue exclusion method after 24 h of treatment. To identify morphological changes induced by extract treatment, cells were examined using a phase contrast microscope. Cells displaying apoptotic changes were identified using previously defined morphological criteria including chromatin condensation, nuclear fragmentation, and blebbing of cytoplasmic membrane.Log phase cell suspension of all cell lines at a concentration of 105 cells/well in 96-microtitre plate. The cells were treated with different concentration of test extract  against the apoptosis inducers as a positive controls  and the negative controls which contained only the medium and incubated for 24 h at 37°C. MTT  solution was added to each well to obtain the final concentration of 400 μg/mL and further incubated at 37°C in a CO2 incubator for 3 h. The reaction resulted in the reduction of MTT by the mitochondrial dehydrogenase of viable cells to a purple formazan product. The MTT-formazan product was dissolved in DMSO and estimated by measuring the absorbance at 570 nm in an ELISA plate reader.Cell lines at exponential growth phase were washed, trypsinized, and resuspended in fresh medium. Cells were kept at a concentration of 102) for 6 h with the nonradioactive thymidine analogue BrdU, which is incorporated into the genomic DNA. Plates were centrifuged 10 min at 250 ×g. Supernatants were removed and 100 μL of lysing solution was added to wells and incubated for 30 min at room temperature. Plates were centrifuged and BrdU-labeled DNA fragments released from the cells into the cell cytoplasm during apoptosis were detected immunologically by using an anti-DNA antibody bound to the microtitre plate to capture the DNA fragments. After an incubation of 90 min at room temperature with anti-BrdU-antibody peroxidase-conjugate, plates were washed, 100 μL of substrate solution was added to each well, and plates were read in an ELISA reader at 450 nm.Apoptosis was determined by using a commercial cellular DNA fragmentation ELISA, a photometric enzyme-linked immunosorbent assay for the detection of BrdU-labeled DNA fragments in culture supernatants and cell lysates. Cells proliferating in vitro were incubated at 37°C in a humidified atmosphere ; Bcl-2 ; Bcl-x ; Bax . Real time PCR reactions were performed using SybrGreen master mix in a 7500 real time PCR system . 35 cycles were carried out, each consisting of denaturation at 95°C for 30 sec, annealing for 30 sec at 60°C, and extension at 72°C for 30 sec. Extension during the final cycle continued at 72°C for 10 min. The resulting amplified fragments were identified on 1.5% agarose gel electrophoresis by ethidium bromide staining and the intensity of bands was quantified by densitometry. To calculate the changes in mRNA expression, we first normalized mRNA expression of target gene to the β-globin mRNA expression in a given sample; the mRNA expression for each gene in the experimental treatment was compared with the level of mRNA expression in the nontreated cells.Total RNAs were isolated from SW872, SW982, HS 39.T, HS 5.T, HL-60, M14WM, MCF-7, and HT29 cell lines treated withn method . RNA wasp < 0.05.Data were analyzed using SPSS 12.0 software for Windows. The experimental data were expressed as mean ± SD, the significance of difference among the various treated groups and positive and negative control groups was analyzed by means of one-way ANOVA, and the level of significance was set at μg/mL concentration were Illicium verum, Glycyrrhiza glabra, Rhamnus Frangula, and Linum usitatissimum. Their levels of growth inhibition occurred at very similar values with regard to all the different cell lines. A weaker activity was observed for the Linum usitatissimum, which showed only a 25% of inhibition toward the SW872 cell line. The lowest in vitro inhibitory activity was observed for the Mentha piperita, Chamaemelum nobile, Melissa officinalis, Thymus vulgaris, Plantago lanceolata, Calendula officinalis, Tilia europaea, Aloysia citrodora, Syzygium aromaticum, Coriandrum sativum, Cinnamomum verum, Papaver rhoeas, Aframomum, Helianthus annuus, Cuminum cyminum L., Sesamum indicum, Coffea arabica, Curcuma longa, Equisetum arvense, Hypericum perforatum, Origanum vulgare, Rosmarinus officinalis, Camellia sinensis, and Vaccinium myrtillus plant extracts, all being inactive as antiproliferative agent toward the human cell lines  were tested at lower concentration, 10 and 25 μg/mL, respectively. Data are reported in Tables  Linum usitatissimum exhibited, at 10 μg/mL, high levels of cell growth inhibition toward almost all cell lines. Both Illicium verum and Glycyrrhiza glabra showed low levels of activity toward the SW872, SW982, HS 5.T, and HL-60 cell lines.To demonstrate the potential biological activity of alcoholic extracts, we carried out a screening of the extracts by measuring their in vitro antiproliferative properties against several common human cancer cell lines. Some plant extracts exhibited antiproliferative activity toward all the cancer cell lines. The cellular growth inhibition percentages for the positive extracts are reported in ll lines . The fou Illicium verum, Glycyrrhiza glabra, Rhamnus Frangula, and Linum usitatissimum at a concentration of 25 μg/mL. Representative results are shown in  Illicium verum show signs of apoptosis .Drastic morphological changes, suggesting induction of apoptosis, were observed after treatment with Illicium verum, Glycyrrhiza glabra, Rhamnus Frangula, and Linum usitatissimum at a concentration of 10, 25, and 50 μg/mL, as compared to negative controls .The major biochemical hallmark of apoptotic cell death is the cleavage of chromosomal DNA at internucleosomal sites into fragments or multiple of about 200 bp. DNA isolated from SW872, SW982, HS 39.T, HS 5.T, HL-60, M14WM, MCF-7, and HT29 cell lines treated withμg/mL of Illicium verum, Glycyrrhiza glabra, Rhamnus Frangula, and Linum usitatissimum. The percentage of positive mRNA expression was variable for different cell lines, being the highest positive cell lines observed in the group treated with Glycyrrhiza glabra and Rhamnus Frangula. Conversely, the expression of both Bcl-2 and Bcl-XL was almost negative in all cell lines treated with Illicium verum, Glycyrrhiza glabra, Rhamnus Frangula, and Linum usitatissimum extracts  is the starting material of Tamiflu® (Roche Laboratories), an antiviral drug which has gained popularity with the recent spread of the bird flu (H5N1). In addition anise extract typically contains 1% to 3% volatile anise oil. The primary constituent of anise oil is anethole (80% to 90%). Other components include alpha-pinene, linalool, anisaldehyde, and methyl chavicol. The composition of anise oil from Illicium verum resembles that of anise oil obtained from Pimpinella anisum but also contains trace quantities of safrole and myristicin [Shikimic acid extracted from the pods (which wraps the seeds) of Chinese star aniseristicin .Glycyrrhiza glabra (licorice) has a long history of medicinal use in Europe and Asia. At high doses, it shows potentially severe side effects, including hypertension (high blood pressure), hypokalemia (low blood potassium levels), and fluid retention. Most adverse effects have been attributed to triterpene saponins . The other compounds vary from species to species and depend on the provenance of the plant. Several flavonoids, as well as other phenolic constituents, are found in licorice; amines, amino acids, sterols, sugars, and starch are also present [ G. glabra has shown considerable antioxidant activity and protective effect against the human lipoprotein oxidative system. present  and etha Rhamnus Frangula, guided by assays for tumor-inhibitory activity, led to the isolation of aloe emodin [A systematic fractionation of an ethanol-water (1 : 1) extract of the seeds ofe emodin . This coe emodin .Linum usitatissimum is a rich source of the essential fatty acid alpha-linolenic acid, which is a biologic precursor to omega-3 fatty acids such as eicosapentaenoic acid. Although omega-3 fatty acids have been associated with improved cardiovascular outcomes, evidence from human trials is mixed regarding the efficacy of flaxseed products for coronary artery disease or hyperlipidemia. The lignan constituents of flaxseed (not flaxseed oil) possess in vitro antioxidant and possible estrogen receptor agonist/antagonist properties, prompting theories of efficacy for the treatment of breast cancer. Illicium verum, Glycyrrhiza glabra, Rhamnus Frangula, and Linum usitatissimum are able to induce growth inhibition and apoptosis and modulate the expression of both Bax and p53 proapoptotic genes and the extent of gene expression was obtained at only 10 μg/mL cell culture treatment. Although with some differences, all tumor cell lines showed similar results, being the highest regulation in gene expression observed in SW 982 and SW 872 cell lines with low differences between the four alcoholic extracts.The results obtained in this study suggest that alcoholic extracts from Glycyrrhiza glabra to induce apoptosis in HT-29 cells and confirmed its anticancer property [ Linum usitatissimum containing mainly sterols and triterpenes (21.4%), free fatty acids (17.8%), lignin dimers (12.2%), and lipids (7.7%) showed significant cell lethality and suppression of cell vitality and proliferation of tumor cell lines [These results are in line with previous reports that suggested a possible use for the management of metastatic malignant tumors . In a reproperty . In addill lines .Many advances in the management of cancer have been achieved since those early times when cancer chemotherapy started to take shape with the discovery of antitumor activity of alkylating agents  and certThe advances in cancer chemotherapy were also greatly aided by progress made in diagnostic procedures, by the advent of combined modalities of treatment, and, last but not least, by the development of improved criteria for regimen design and result assessment . As a coDespite this progress, major difficulties remain to be overcome before cancer therapeutics can become generally successful in the curative management of cancer. This is particularly so in the case of the common so-called solid tumors. These difficulties can be attributed for the most part to the lack of agents acting uniquely and specifically on tumors, or at least having sufficiently marked selectivity of antitumor action, and to the phenomenon of resistance.These two limitations combined are the main reasons for ultimate failure: in fact, even a minor level of cellular resistance may be sufficient to impart clinical resistance because one cannot overcome it by increasing adequately drug dose intensity without incurring nonacceptable toxicity. During the past two decades new vistas have been opening up in cancer therapeutics consequent to progress made in the understanding of the molecular biology of the cancer cell, of the interaction between tumor and host regulatory mechanisms, and of the mechanisms responsible for different forms of resistance.The main purpose of this study was to evaluate the induction of apoptosis in human cell lines treated with alcoholic herbal extracts. Apoptosis is a selective physiologic deletion of cells that are no longer required. The concept of apoptosis could be of great importance for cancer treatment since the internal cellular program of cells to commit suicide can be initiated by cell damaging agents like antineoplastic drugs as well as by natural derived compounds. Some researchers have postulated that the effect of some antineoplastic compounds is caused by apoptosis-induction. It has been found by several authors that inhibition of apoptosis induces resistance to chemotherapeutic drugs . Bax andAlthough much remains to be learned in each of antitumor research areas that no doubt will have an impact on the management of cancer, it is already possible to identify new directions in cancer therapeutics aimed at exploiting the knowledge acquired so far. The approaches that are being pursued at present are essentially as follows: (1) the development of specific or highly selective antitumor agents affecting newly discovered cellular sites of potential intervention; (2) the increase of antitumor effectiveness of available drugs through the design of improved treatments, including combination treatments based on the knowledge of the mechanisms of antitumor and toxic drug action; (3) the modification or prevention of resistance to individual or multiple drugs, whether natural or acquired, through the alteration of relevant gene expression and/or the modification of biochemical mechanisms involved; (4) the induction of therapeutically profitable alterations of biological responses to tumor through the administration of soluble or cellular effectors, or their blockage and/or modulation; (5) the development of combined treatment modalities maximizing antitumor action and taking advantage of differences in the limiting toxicities of the treatments combined; and (6) the development of treatments tailored to individual patients based on improved assessments of potential tumor responsiveness and of the limitations related to the heterogeneity of tumor cell populations. Illicium verum, Glycyrrhiza glabra, Rhamnus Frangula, and Linum usitatissimum have shown significant reduction in in vitro tumor cell proliferation. Additional studies are needed to identify how these natural extracts could be used as a complementary approach to currently used chemotherapies for different cancers. Also, more studies designed to investigate the molecular mechanisms underlying anticancer activity are needed. Altogether, natural extract use holds promise as an adjuvant treatment to prevent tumor cell growth.In summary, extracts of


The molecular background of the enhanced glucose uptake and its regulation in lymphoma cells is not fully clarified and may provide new possibilities to reverse the altered metabolism. Thus in this study we investigated regulation of glucose uptake by different signaling pathways. Furthermore, the effect of the glucose analog 2-deoxy-d-glucose (2-DG) alone and in combination with other inhibitors on cell survival was studied.Changes in glucose and energy metabolism contribute to the altered phenotype of cancer cells and are the basis for positron emission tomography with An FDG uptake assay was established and uptake of FDG by lymphoma cells was determined after incubation with inhibitors of the c-MYC and the PI3K signalling pathways that are known to be activated in lymphoma cells and able to regulate glucose metabolism. Inhibitors of MAPK signalling pathways whose role in altered metabolism is still unclear were also investigated. Expression of mRNAs of the glucose transporter 1 (GLUT1), hexokinase 2 (HK2), glucose-6-phosphatase (G6Pase) and lactate dehydrogenase A (LDHA) and of the glucose metabolism-regulating micro RNAs (miRNA) miR21, -23a, -133a, -133b, -138-1 and -143 was determined by RT-PCR. Cell viability was analysed by MTT assay.Treatment with the c-MYC inhibitor 10058-F4 and inhibitors of the PI3K/mTOR pathway diminished uptake of FDG in all three cell lines, while inhibition of MAPK pathways had no effect on glucose uptake. Expression of glycolysis-related genes and miRNAs were diminished, although to a variable degree in the three cell lines. The c-MYC inhibitor, the PI3K inhibitor LY294002, the mTOR inhibitor Rapamycin and 2-DG all diminished the number of viable cells. Interestingly, in combination with 2-DG, the c-MYC inhibitor, LY294002 and the p38 MAPK inhibitor SB203580 had synergistic effects on cell viability in all three cell lines.c-MYC- and PI3K/mTOR-inhibitors decreased viability of the lymphoma cells and led to decreased glucose uptake, expression of glycolysis-associated genes, and glucose metabolism-regulating miRNAs. Inhibition of HK by 2-DG reduced cell numbers as a single agent and synergistically with inhibitors of other intracellular pathways. Thus, targeted inhibition of the pathways investigated here could be a strategy to suppress the glycolytic phenotype of lymphoma cells and reduce proliferation. Cancer cells on the other hand take up larger amounts of glucose compared normal cells and metabolize it through glycolysis at higher rates , stabilization of transcription factors like hypoxia-inducible factor 1 (HIF-1) and inactivation of tumor suppressor genes like p53 , 9. ConcNon-Hodgkin’s lymphoma (NHL) is a heterogeneous group of diseases and includes several lymphoma subtypes like diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, mantle cell lymphoma, follicular lymphoma and others . NHLs diThe involvement of PI3K, c-MYC and MAPK pathways on glucose uptake, expression of glycolysis-associated genes and cell proliferation in lymphoma is not known yet.d-glucose (2-DG), a glucose analogue that decreases glycolytic rate by inhibiting HK, as a single agent and in combination with inhibitors of the c-MYC-, PI3K-, and MAPK signaling pathway inhibitors on cell viability.Thus in this study we performed experiments on the regulation of glucose uptake, transcription of genes involved in glycolysis, and regulation of miRNAs by the PI3K/AKT/mTOR pathway, c-MYC and different MAPK signaling pathways. Moreover, we studied the effect of 2-deoxy-2 were from Sigma Aldrich . They were stored in 10 mM aliquots in DMSO at −20 °C and further diluted in the appropriate medium. 2-deoxy-d-glucose (2-DG) was from Sigma-Aldrich and was dissolved in water and stored as 1 M aliquots at −20 °C.The c-MYC-Inhibitor 10058-F4 , the p382.Established human lymphoma cell lines BJAB, OCI-LY3 and SU-DHL-6 were used in this study. BJAB cells were derived from a Burkitt lymphoma, SU-DHL-6 cells from a follicular B cell lymphoma and OCI-LY3 cells from a DLBCL. The BJAB and SU-DHL-6 cell lines were classified as germinal center B cell like (GCB), OCI-LY3 cells were classified as activated B cell like (ABC) . Cell li5 cells were seeded into each well of a 24 well plate in their appropriate medium containing 0.5% FBS and the inhibitors and concentrations indicated. After incubation for 24 h, cells were harvested by centrifugation and resuspended in glucose-free RPMI medium (Thermo Fisher) supplemented with 5.5 mM glucose  −1) * 100 [To ensure similar PCR efficiencies as a prerequisite for relative quantification with the ΔΔCT method, PCR efficiency of each primer and probe set was first determined by measuring serial tenfold dilutions of two templates. PCR efficiency was calculated according to the equation: PCR efficacy = (10 1) * 100 . All PCRWe first analyzed mRNA expression of the GLUT1 gene after incubation with the five inhibitors. Incubation with the c-MYC inhibitor and the mTOR inhibitor Rapamycin after 24 h led to a significant down-regulation of GLUT1 mRNA in all three cell lines  expression was significantly decreased by the c-MYC inhibitor, Rapamycin and LY294002 in all three cell lines Table . In SU-DGlucose-6-phosphatase (G6Pase) expression was found to be positively regulated by the c-MYC inhibitor, Rapamycin and LY294002 in all three cell lines. PD98059 incubation led to an increase only in BJAB cells, while G6Pase mRNA remained unchanged after this treatment in SU-DHL-6 and OCI-LY3 cells. SB203580 incubation did not affect G6Pase mRNA expression in all three cell lines Table .miRNA21 expression was diminished by the c-MYC inhibitor and Rapamycin in all three cell lines, while LY294002 incubation led to a decrease in miRNA21 only in BJAB and OCI-LY3 cells  , 34. In Treatment of our NHL cell lines with the PI3K inhibitor LY294002 or the mTOR inhibitor Rapamycin led to a decrease in cell viability in all three cell lines with IC50 values in the range of around 1 µM for Rapamycin and 10 µM for LY294002 (Fig. In addition to the effects on the number of viable cells, both inhibitors led to a decreased FDG uptake after 24 h treatment as expected Fig. . A similUnexpectedly, while Rapamycin led to a decrease in GLUT1 mRNA expression, treatment with LY294002 did not significantly affect GLUT1 mRNA in all three cell lines (Table The decrease of LDHA mRNA expression as well as the increase in G6Ptase after incubation with LY294002 and Rapamycin in all three cell lines corresponds to a diminished glycolysis after PI3K/AKT/mTOR inhibition, since LDHA is a glycolytic enzyme while G6Ptase catalyzes the reaction from glucose-6-phosphate back to glucose [HK2 mRNA expression was diminished in all three cell lines after incubation with Rapamycin, but not with LY294002. This may reflect the distinct levels of inhibition by the two substances. LY294002 inhibits PI3K, while Rapamycin inhibits mTOR proteins that are located downstream of PI3K and AKT. Treatment with LY294002 may therefore be circumvented by activation of the downstream AKT and mTOR kinases by alternative pathways or mutations and thus may explain the different effects of LY294002 and Rapamycin in our cells .The changes in miRNA expression after treatment with LY294002 or Rapamycin primarily reflect the oncogenic features of the PI3K/mTOR pathway in NHL cells with downregulation of the oncogenic miRNA21 and upregulation of the tumor suppressor miRNAs 133a, -133b and -138-1 (Table c-MYC is an oncogenic transcription factor that is overexpressed in many cancer types including B- and T-cell malignancies and is involved in cell metabolism and cell proliferation . In lympIncubation of our cells with the c-MYC inhibitor as expected led to a decreased number of viable cells Fig. a with ICBesides a reduction of the number of viable cells, treatment of lymphoma cells with the c-MYC inhibitor led to a marked decrease in FDG uptake in all three cell lines with the SU-DHL-6 cells showing the most pronounced effect Fig. . CorrespAs expected, expression of the mRNA encoding LDHA was also diminished after incubation with the c-MYC-inhibitor Table  while exThe changes in miRNA expression after c-MYC inhibition resemble those after inhibition of PI3K/mTOR probably reflecting the function of c-MYC as an oncogene in NHL cells. In contrast to the results obtained after mTOR inhibition with Rapamycin, after treatment with the c-MYC inhibitor or LY294002, the expression of the miRNA133a was diminished (Table The p38-MAPK signaling pathway is involved in many cellular functions like differentiation, proliferation and induction of cell death. The exact role of p38-MAPK in a cancer cell depends on the cell type and the tumor stage . In NHL,MEK1 gene of 43% [The MEK pathways are activated mainly in response to the stimulation of tyrosine kinase receptors . MEK pate of 43% . The datThe glucose analog 2-DG is an inhibitor of HK that is used to block the Warburg effect in cancer cells . 2-DG isDue to its ability to inhibit glycolysis, 2-DG has been evaluated as an anticancer agent in several cell systems Review: . In our Although 2-DG decreases the number of viable cells in short-time cell culture experiments, it has not been effective as a single agent in vivo . We therA synergistic effect of a combined treatment with 2-DG and PI3K/mTOR inhibitors as found in our experiments has already been described by a few authors: In lung cancer cell lines, an analogue of Rapamycin hypersensitized cells to 2-DG treatment under hypoxic conditions . FurtherCombined treatment with 2-DG and SB203580 has recently been described in pancreatic and ovarian cell lines  with simUp to now, to the best of our knowledge, no data on the efficacy of a combined treatment with 2-DG and the c-MYC inhibitor or MEK inhibitor like PD98059 are available. Inhibition of c-MYC alone resulted in significant decrease of viable cells which was synergistically enhanced by 2-DG in our cells Table . On the Taken together, our results suggest that PI3K/mTOR inhibitors as well as the c-MYC inhibitor decreased the viability of lymphoma cells, reduced glucose uptake and glucose metabolism, and downregulated the expression of glycolysis-associated genes and glucose metabolism-regulating miRNAs. Our results that 2-DG decreased viability of all cell lines support the hypothesis that lymphoma cells are highly dependent upon high glycolysis for survival. In combination with inhibitors of intracellular signaling pathways, 2-DG treatment may be an option to reduce proliferation of NHL cells.


Purpose: Study on gold based therapeutic agents for cancer cells deracination has become under scrutiny in recent years owing to effective treatments are not available for rapidly progressive cancers. The aim of present study was to examine efficiency of radioactive 198Au/PAMAMG4 and non-radioactive 197Au/PAMAMG4 nancomposites against 4T1 and MCF7 breast cancer cell lines.Methods: The PAMAMG4 dendrimer was treated with the gold anions and then, the mixture was chemically reduced by NaBH4. Prepared 197Au/PAMAMG4 was bombarded by thermal neutrons in the Tehran Research Reactor to 198Au/PAMAMG4 be produced. Prepared nanocomposites were characterized by means of FT-IR, 1H NMR, Zeta-potential measurements, TEM and EDX analyses. The radionuclidic purity of the 198Au/PAMAMG4 solution was determined using purity germanium (HPGe) spectroscopy and its stability in the presence of human serum was studied. In vitro studies were carried out to compare toxicity of PAMAMG4, 197Au/PAMAMG4 and 198Au/PAMAMG4 towards 4T1 and MCF7 cancerous cells and C2C12 normal cell lines.Results: Characterization results exhibited that invitro agents, 197Au/PAMAMG4 and 198Au/PAMAMG4, were synthesized successfully. Cells viability after 24 h, 48 h, and 72 h incubation, using MTT assay showed that the toxicity of 198Au/PAMAMG4 is significantly superior in comparison with 197Au/PAMAMG4 and PAMAMG4. Furthermore, the toxicity of 198Au/PAMAMG4 was higher on cancerous cells especially in higher level of concentrations after 72 hours (P<0.05).Conclusion: In the current study, the preparation of 197Au/PAMAMG4 and 198Au/PAMAMG4 is described and the cytotoxic properties of them against the MCF7, 4T1 cancerous cells and C2C12 normal cells were evaluated using MTT assay. Additionally, the size of the colloids is a function of dendrimer generation.8 As a new class of highly branched, monodispersed, and synthetic macromolecules, poly amidoamine (PAMAM) dendrimers have attracted a great deal of interest in the development of various biological applications.14 The unique structural characteristics of PAMAM dendrimers allow one to design various dendrimer-metal nanocomposite.14 Recently, applicability of PAMAM to deliver anticancer drugs and other bioactives is well-reported in the literatures, especially dendrimers belonging to the fourth (G4) and fifth (G5) generation of PAMAM class.17 Poly amidoamine dendrimers contain a variety of functional groups capable of complexing metal cations, as well as interior voids in their structure capable of hosting small metal components and protecting them from further aggregation.18Gold nanoparticles (NPs) have recently received great technological and scientific interest due to their extensive applications in biology, catalysis, and nanotechnology.19 So far, many of the studies on AuNPs suggest that cancer cells are especially vulnerable to these particles. Thus, AuNP-based treatment can destroy cancer cells, with minimal injury to healthy cells.20The development of AuNP-based strategies for the eradication of cancer cells is important, because effective therapies are frequently not available for rapidly progressing cancers.21 4T1 breast carcinoma is a highly malignant and poorly immunogenic marine tumor model that resembles advanced breast cancer in humans, and is refractory to most immune stimulation-based treatments.22Breast cancer, which is most resulted in metastasis, is the leading cause of cancer death in women with more than a million newly diagnosed cases annually worldwide.24 Prior to MCF7, it was not possible for cancer researchers to obtain a mammary cell line that was capable of living longer than a few months.MCF7 is a breast cancer cell line isolated in 1970 from a 69-year-old Caucasian woman. MCF7 is the acronym of Michigan Cancer Foundation-7, referring to the institute in Detroit where the cell line was established in 1973 by Herbert Soule and co-workers.25Cytotoxicity assay is an appropriate method for screening new drugs within a short time in order to determine cell killing property of these chemical compounds regardless the mechanism of cell death. Usually in oncology research and clinical practices, in vitro testing is preferred prior to in vivo studies. MTT assay has been described as rapid, simple and reproducible method, widely used in the screening of anticancer drugs and to measure the cytotoxic properties.197Au/PAMAMG4 and 198Au/PAMAMG4 is described and the cytotoxic properties of them against the MCF7, 4T1 cancerous cells and C2C12 normal cells were evaluated using MTT assay.In the current study, the preparation of 4. 3H2O), sodium borohydride (NaBH4), sodium hydroxide, DMSO (dimethyl sulfoxide) and Whatman paper were purchased from (Merck. Germany). RPMI medium 1640, DMEM medium, fetal bovine serum (FBS), gentamicin, streptomycin, penicillin G, trypsin, EDTA were obtained from . MCF7 human breast cancer cells, 4T1 mice breast adenocarcinoma cells, and C2C12 mice muscle normal cell were obtained from National Cell Bank of .Materials used in this study included methanolic solution of PAMAMG4 dendrimer and MMT -2,5-diphenyltetrazolium bromide) obtained from . Hydrogen tetracholoroaurate .28 Firstly 0.5μmol dendrimer was added to 5 mL of water with vigorously stirring. Then 5 mL of an aqueous solution of 0.5 μmol HAuCl4 was added to the above solution. After stirring for further 45 min, procedure followed by addition of a basic aqueous solution of sodium borohydride (25 mM NaBH4 in 0.3 M NaOH). The prepared light yellow solution turned to brown, indicating the formation of colloidal gold. The stirring process was continued for 1 h to complete the reaction. The reaction mixture was then dialyzed against PBS  and water  for 1 day to remove the excess reactants. At this stage 197Au/PAMAMG4 was prepared.197Au captures neutrons very efficiently because of its large cross section. 198Au (t1/2=2.69 days) decays dominantly by beta-radiation  and with a small gamma component .29 Irradiation was performed at Tehran Research Reactor (TRR). Sample was irradiated in quartz vials for 2 hours, in core-face location, with a thermal neutron flux of 1 × 1011ncm-2s-1. The radionuclidic purity of the prepared 198Au/PAMAMG4 solution was determined for the presence of other radionuclides using purity germanium (HPGe) spectroscopy, through the detection of various interfering gamma emitting radionuclides. Final 198Au/PAMAMG4 solution  was incubated in the presence of freshly prepared human serum (300 µL)  at 37°C for 7 days. The stability study of 198Au/PAMAMG4 in final formulation was performing ITLC analysis using Whatman chromatography paper eluted with methanol: water (7:3) mixture. 2O as a solvent and Tetramethylsilane (TMS) as an internal standard. The morphology of prepared nanocomposites was studied by transmission electron microscopy  and the content of Au in 198Au/PAMAMG4 was determined by means of energy dispersive X-ray spectrometer . EDX analysis was performed using lyophilized nanocopmosite sample without gold coating.Spectroscopic analyses of the samples were performed using a Fourier transform infrared (FT-IR) spectrometer (Perkin–Elmer 843). Zeta-potential measurements were performed using  with a red diode laser at 633 nm. Proton NMR spectra were conducted on a nuclear magnetic resonance spectrometer  using commercially available D2. The cytotoxicity of the PAMAMG4, 197Au/PAMAMG4 and 198Au/PAMAMG4 were investigated by an MTT cell viability assay. MCF7, 4T1 and C2C12 cells were seeded onto a 96-well plate at a density of 4× 103 cells per well and cultivated in 100 μL of their mentioned medium under 37°C and 5% CO2 for 24 h .30 After that, the mediums were replaced with fresh RPMI and DMEM containing PAMAMG4, 197Au/PAMAMG4 or 198Au/PAMAMG4 with different concentrations ranging from 50 nM to 400 nM and the cells were incubated under 37 °C and 5% CO2. The MTT assay was applied to study the cell viability after 24 h, 48 h and 72 h of drugstreatment. MTT  was added into each sample and the cells were incubated at 37 °C and 5% CO2 for 4 h. Thereafter, the medium was removed and 150μL dimethylsulfoxide (DMSO) was added to dissolve the formazan crystals. The assays were performed according to the manufacturer’s instructions using a Biochrom Scientific Anthos 2020 ELISA reader  at 570 nm.32The MCF7 human breast cancer cells, 4T1 mice breast adenocarcinoma cells and C2C12 mice muscle normal cells were regularly cultured in RPMI 1640 medium and DMEM medium, respectively, supplemented with 10% FBS  and 1% penicillin-streptomycin at 37°C and 5% COAll the experiments were repeated at least three times. Results are presented as the mean ±SD. Statistical comparisons were carried out using a three and four way analysis of variance ANOVA with post hoc testing for comparison of Sidak tests. Pair-wise comparisons between treatments were made using independent sample t-test . A p-Value of < 0.05 was considered statistically significant.197Au/PAMAMG4 and 198Au/PAMAMG4 nanocomposites was studied by microscopic visualization. The morphology of the cells was observed using an invert optical microscope  under a magnification of 200×.The changes of 4T1 cell morphology after treatment with the 197Au/PAMAMG4 and 198Au/PAMAMG4 nanocomposites. The absorption peak at 3353cm-1 corresponded to N–H deformation vibration of tertiary amide, while the peak centered at 3287cm-1 was assigned to the N–H stretching vibration arising from its association. The next two peaks at 2944cm-1 and 2844cm-1 indicated unsymmetrical and symmetrical stretching vibration of methylene.33 The amide band present at 1640cm-1 is characteristic of the dendrimer branches.8 Also, N-H bending exhibits broad band at about 1640cm-1 for primary amine. The stretching vibration of C-N bonds is appeared at 1368cm-1. The two peaks at 1132cm-1 and 1202cm-1 were related to the stretching vibrations of tertiary amine and primary amine, respectively. N-H out of plane bending occurs at 800cm-1and the peak at 550cm-1 showed the bending vibration of tertiary amine.34 Comparison of FTIR curve of non-active 197Au/PAMAMG4 compound with radioactive 198Au/PAMAMG4 exhibited that the N-H band present at 1640cm-1 is stronger at non-active complex. Furthermore, the intensity of the peak assigned at 1368cm-1 is increased in the radioactive complex. It could be due to the cleavage of some N-H bands of primary amines and production of some new tertiary amines as a result of polymerization of nanocomposite particles under described neutron bombarding condition. Partial cross-linking of the organic component (radiation polymerization) was also reported by Khan and co-workers.30 Moreover, careful comparison of two curves reveals that neutron bombarding of 197Au/PAMAMG4 induced a small shift in the stretching vibration of C-N bond of prepared 198Au/PAMAMG4 to higher energy.1H Nuclear Magnetic Resonance spectra of 197Au/PAMAMG4 and 198Au/PAMAMG4 nanocomposites are shown in 1H NMR spectrum of 198Au/PAMAMG4 nanocomposite indicate partially of polimerization crosslinked PAMAMG4 during the neutron irradiation. The exact mechanism of polimerization is not known up to now. According to Tang and co-workers reports, the radical mechanisms and the local heat effects causing beta-alanine crosslinking, and in retro-Michael reaction may be responsible to take place a rearrangement of the molecular structure and finally polymerization.35197Au/PAMAMG4 and 198Au/PAMAMG4 were also carried out to investigate the surface potential changes after the neutron irradiation. As shown in 197Au/PAMAMG4 (-23.9 mV) significantly changed after the neutron bombarding and producing of 198Au/PAMAMG4 (-37.6 mV). Decreasing of surface positive charge could be related to involvement of primary amine terminal groups in the ensuing polymerization reactions during neutron bombardment.36Zeta-potential measurements for 197Au/PAMAMG4 and 198Au/PAMAMG4 compounds. Comparision of two pictures reveals that 198Au/PAMAMG4 has larger particle size compared to 197Au/PAMAMG4. This could be related to partial polymerization of PAMAMG4 due to neutron bombardment.29Transmission electron microscopy analyses of nanoparticles were performed to understand the morphology and size of particles. TEM images  show spherical shape for 198Au/PAMAMG4 nanocomposie was checked by gamma-ray emission spectroscopy. Two main radioisotopes of gold have charming nuclear properties that make them desirable for imaging and therapy purposes. 198Au has a moderate-energy beta maximum (0.96 keV) making it a good candidate for therapy, and a gamma emission (411 keV) that allows for in vivo tracking and dosimetry calculation. These properties led to the medical use of 198Au as a brachytherapy agent for breast cancer. A second radioisotope, 199Au, has a low-energy beta maximum and emits a 158keV gamma that is easily detected by SPECT camera.37198Au (t1/2 = 2.69 days), and there is no significant peak related to the 199Au isotope in the HPGe spectrum.The radionuclidic purity of the 198Au/PAMAMG4 at pH=7.5. The 198Au/PAMAMG4 complex in presence of human serum at 37 °C was found to be stable in final pharmaceutical sample and its radiochemical purity was calculated to be above 93.5% a week after the preparation by using Whatman 3 MM paper eluted with methanol: water (7:3) mixture.198Au/PAMAMG4 and non- radioactive 197Au/PAMAMG4 against MCF7, 4T1 and C2C12 cells were assessed using the MTT assay. The MTT assay is based on the ability of a mitochondrial dehydrogenation enzyme in viable cells to cleave the tetrazolium rings of the pale yellow MTT and form formazan crystals with a purple color.38 Therefore; the number of surviving cells is directly proportionate to the level of the formed formazan. Figures 7a, b and c reveal the cell viability of the cultured MCF7, 4T1 and C2C12 cells treated by 50, 100, 200 and 400 nM of PAMAMG4,197Au/PAMAMG4 and 198Au/PAMAMG4 solutions. Control groups use only medium.The cytotoxicity of the radioactive 197Au/PAMAMG4 and 198Au/PAMAMG4 on viability of three cell lines of MCF7, 4T1 and C2C12 in various concentrations after 24h incubation, based on the results of three way ANOVA showed a three way interactions of cell lines* drugs *concentrations as (P<0.001).Assessing the effect of drugs including PAMAMG4, 197Au/PAMAMG4 and 198Au/PAMAMG4 on viability of each of the cell lines varies as a function of the concentration of drugs.The result of all two ways interactions are as follow: cell lines* drugs (P<0.001), cell lines*concentrations (P<0.001), and drugs *concentrations (P<0.001), and for all main effects of cell lines, drugs and concentrations (P<0.001) is obtained. This means that the effect of PAMAMG4, 198Au/PAMAMG4*concentration 400 (C400). In the other words, after 24 hours, treatment of MCF7 and 4T1 cell line with 198Au/PAMAMG4 leads to lowest amount of viability in the highest level of concentration .For all two ways interactions cell lines* drugs, cell lines*concentrations and drugs*concentrations P is lower than 0.001. Also for all main effects of cell lines, drugs and concentrations P are lower than 0.001.198Au/PAMAMG4 * and C2C12*198Au/PAMAMG4*(C50) levels. The other levels of three factors were significantly in higher amounts of viability. Hence, after 48 hours, 198Au/PAMAMG4 of MCF7 and 4T1 cell lines lead in lower amount of viability in the higher level of concentration  levels, and other levels of the three factors had significantly higher amount of viability. As shown in 198Au/PAMAMG4 towards 4T1 cell line results in the lowest amount of viability in higher level of concentration. To compare the changes in the viability during three time points of 24, 48 and 72 hours along with the effect of cell lines, drugs and concentrations, the findings of four way analysis of variance revealed the significant four ways interaction effect of times*cell lines* drugs*concentrations =7.42, P < 0.001). It means that the changes of cell viability during three time points of 24, 48 and 72 hours varies for cell lines, each drug  and within the levels of concentrations (C50 to C400). As can be seen the effect of cell lines, drugs  and concentrations leads in significantly the lowest amount of viability after 72 hours based on the results obtained from the Sidak post hoc tests (P < 0.05).After 72 hours incubation, the results of three way ANOVA to study of the effect of drugs materials on viability of three cell lines of MCF7, 4T1 and C2C12 in various concentrations of drugs showed significant three ways interactions of cell lines* drugs *concentrations (P < 0.001), all two ways interactions of cell lines* drugs (P < 0.001), cell lines*concentrations (P < 0.001), and drugs *concentrations (P < 0.001) as well as for all main effects of cell lines, drugs and concentrations (P < 0.001). In the other words, the effects of drugs including PAMAMG4, 197Au/PAMAMG4 and 198Au/PAMAMG4. It is reported that the treatment of cells with some test compounds induced morphological alterations such as retraction of cytoplasmatic expansions; detachment, formation of the round shaped cells as well as membrane blebs, all suggestive the induction of programmed cell death.39The cytotoxicity effects of the compounds towards the 4T1 cell line were further confirmed by microscopic visualization of changes of the cell morphology after treatment with the 197Au/PAMAMG4 and 198Au/PAMAMG4, after 24 h, respectively. As shown in 197Au/PAMAMG4 nanocomposite with concentration (400 nM) compared with the untreated cells. According to 197Au/PAMAMG4 to some extent. In contrast, 198Au/PAMAMG4 treatment. This phenomenon could be related to apoptosis of 4T1 cells due to 198Au/PAMAMG4 (16.64 μCi) treatments. These results are consistent with the MTT assay data.198Au/PAMAMG4 nanocomposite was prepared successfully by neutron bombardment of synthesized non-radioactive 197Au/PAMAMG4. 1H NMR, FT-IR and TEM analyses reveals partially cross-linked polymerization of PAMAMG4 in the repared neutron bombarded sample (198Au/PAMAMG4). In vitro anti-cancer assessments of radioactive 198Au/PAMAMG4 mater and non-radioactive 197Au/PAMAMG4 with regard to the MCF7, 4T1 and C2C12 cell lines were investigated. Analysis of variance followed by Sidak post-hoc test, shows that the toxicity of 198Au/PAMAMG4 is significantly deferent from 197Au/PAMAMG4 and PAMAMG4 on all of the three cell lines. The toxicity of 198Au/PAMAMG4 is more on cancerous cells compared to normal cells especially in higher level of concentrations after 24, 48 and 72 hours (P<0.05). In conclusion, 198Au/PAMAMG4 inhibited growth of MCF7 and 4T1 breast cancer cells in a dose- and time-dependent manner in vitro. Moreover, the effect of cell lines, drug types and concentrations of drugs leads to the lowest amount of viability after 72 h compared with 24 h and 48 h.In this study, radioactive Support by Nuclear Science and Technology Research Institute of Atomic Energy Organization of Iran and Iran National Science Foundation (INSF) is greatly appreciated.Not applicable.The authors declare no conflict of interests.


The data for each cell line have been presented as raw number of citations, percentage share of the total citations for each search engine and as an average percentage between the three search engines. In order to correct for the time since each cell line has been in use, the raw citation data have also been divided by the number of years since the derivation of each cell line. This is a supporting article for a review of  Specifications TableValue of the data•The data shows the relative popularity for each of the established medulloblastoma cell lines.•The most frequently cited cell lines can be easily seen along with the number of papers where they are mentioned.•In conjunction with the review article, the data can be used to link cell lines and medulloblastoma subtypes.•Researchers can readily identify underrepresented medulloblastoma subtypes, such as WNT and Group 4.•The relative merits of using Google Scholar, Web of Science and Scopus to find information about specific cell lines can be compared.1Data are organised in three spreadsheets within one Excel file. The “Raw Citations” spreadsheet gives the numbers of citations for each cell line as determined by a search of Google Scholar, Web of Science and Scopus databases. It also includes the date of derivation for each cell line and the difference with the current year (2016). The second spreadsheet named “Analysis” includes a calculation of the relative percentage of the citations for each cell line compared to the total number of citations; first for each search engine, and then as a mean of the percentage results for all three search engines. The same procedure is applied to the citation data divided by the number of years since the first publication of the cell line. Cell lines are then ranked for their relative popularity both before and after correcting for the time they have been in use. The ranks have been used to create the pie charts displaying the relative frequency of scientific mentions for the 15 most popular cell lines in Figure 2 of 2Web of Science, Scopus and Google scholar databases were searched using the name of each cell line and the term medulloblastoma. The number of all citations in Google Scholar and the number of original articles  mentioning the keywords were documented for each cell line. Wildcards were used to find citations encompassing the different ways of spelling the cell lines. For example “D$283 and medulloblastoma” to find all mentions of “D-283, D 283 or D283”. The advantage of Web of Science and Scopus are that only original articles can be selected, while Google Scholar includes “grey literature”, such as conference proceedings and scientific posters. Nevertheless, Google Scholar can retrieve citations documenting the use of cell lines from the body of the articles without them necessarily being mentioned in the topic, abstract or keywords.The list of medulloblastoma cell lines was compiled from Cellosaurus, the controlled vocabulary of cell lines developed by the Swiss Institute of Bioinformatics


NF2 tumor suppressor gene, encoding a key regulator of the Hippo signaling pathway, is observed in 22.5% of PRCC. The Hippo signaling pathway controls cell proliferation by regulating the transcriptional activity of Yes-Associated Protein, YAP1. Loss of NF2 results in aberrant YAP1 activation. The Src family kinase member Yes also regulates YAP1 transcriptional activity. This study investigated the importance of YAP and Yes activity in three NF2-deficient PRCC cell lines. NF2-deficency correlated with increased expression of YAP1 transcriptional targets and siRNA-based knockdown of YAP1 and Yes1 downregulated this pathway and dramatically reduced cell viability. Dasatinib and saracatinib have potent inhibitory effects on Yes and treatment with either resulted in downregulation of YAP1 transcription targets, reduced cell viability, and G0-G1 cell cycle arrest. Xenograft models for NF2-deficient PRCC also demonstrated reduced tumor growth in response to dasatinib. Thus, inhibiting Yes and the subsequent transcriptional activity of YAP1 had a substantial anti-tumor cell effect both in vitro and in vivo and may provide a viable therapeutic approach for patients with NF2-deficient PRCC.Papillary renal cell carcinomas (PRCC) are a histologically and genetically heterogeneous group of tumors that represent 15–20% of all kidney neoplasms and may require diverse therapeutic approaches. Alteration of the  MET oncogene and presents with bilateral, multifocal type 1 PRCC [FH tumor suppressor gene and can have an aggressive variant of type 2 PRCC [FH mutation, chromosomal translocations producing TFE3-fusions, or NRF2 pathway mutations in NFE2L2 (NRF2) or CUL3 [MET, SETD2, NF2, KDM6A, and SMARCB1, suggesting that alterations to the MET signaling pathway, Hippo signaling pathway, and the chromatin modifier pathways may play an important role in PRCC carcinogenesis [Papillary renal cell carcinoma (PRCC) is the second most common type of kidney cancer representing approximately 15–20% of all cases. PRCC is heterogeneous and categorized into two main histologic subtypes, type 1 PRCC and type 2 PRCC . HPRC pae 1 PRCC , 5, while 2 PRCC , 7. Althogenesis .NF2) is a tumor suppressor gene that regulates the Hippo signaling pathway, which normally controls organ growth by suppressing cell division and proliferation and activating programmed cell death/apoptosis [NF2 are responsible for the hereditary tumor predisposition syndrome neurofibromatosis type 2, which is characterized by the development of multiple tumors in the nervous system [NF2 gene have been previously reported at a low frequency in a breast cancer, prostate cancer, clear cell renal cell carcinoma, and PRCC [NF2) that induces the MST1/2 complex, with the regulatory protein Salvador, to phosphorylate the LATS1/2/MOB1 complex that leads to the phosphorylation dependent inhibition of the transcriptional activity of Yes-associated protein 1 (YAP1) and transcriptional co-activator with PDZ-binding motif (TAZ) [NF2 gene product Merlin suppresses the transcriptional activity of YAP1 and controls contact inhibition of normal cells. Complete loss of the NF2 gene would lead to an inability to suppress the activity of YAP/TAZ, driving the expression of multiple downstream transcription targets, including BIRC5 (survivin), CTGF (connective tissue growth factor), and CCND1 (cyclin D1), that all promote cell growth and survival [BIRC5 [Neurofibromatosis type 2  . Thus, tsurvival . Aberranl [BIRC5 . Yes inhl [BIRC5 , have bel [BIRC5 , 16 and NF2-deficient PRCC cell lines of targeting YAP1 transcription activation resulting from the Hippo signaling pathway loss. We investigated three PRCC cell lines bearing NF2 mutations and assessed the effect of Yes inhibitors on their proliferation and survival in vitro and in vivo with the goal of developing a novel potential therapeutic approach for PRCC patients whose tumors are characterized by an NF2 deficiency.In this report, we investigated the effect on the growth and survival of NF2 is among the five most commonly mutated genes in PRCC and was present in 3.6% of cases  data has recently shown that s Figure . While tNF2 gene. This identified one cell line derived from a patient with type 1 PRCC, UOK342, with a homozygous NF2 insertion/deletion alteration that resulted in frameshift mutation and the loss of a splice site   Figure , for furNF2 alteration leads to the activation of the transcription factor YAP1 and the expression of multiple downstream targets promoting tumor cell survival and proliferation [NF2 was found in UOK275, UOK342 and ACHN, we investigated whether these alterations led to the expression of YAP1 downstream targets. As shown on Figure NF2-deficient PRCC cells and HEK293 cells using a high content imaging system . As shown in Figure NF2-deficient PRCC cell lines compared to HEK293 cells suggesting that YAP1 is more activated in NF2-deficient PRCC cell lines  and Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL) –22. SaraC Figure  as well C Figure . The embC Figure . To distC Figure . The levNF2 and YAP1 activation lead to aberrant cell proliferation and loss of contact inhibition [NF2-deficient cells were treated with either 50 nM or 500 nM of dasatinib or 100 nM of saracatinib, stained with BrdU and propidium iodide and analyzed by flow cytometry. As shown in Figure NF2-deficient cell lines but did not affect the cell cycle of HEK293 . Female nude mice bearing UOK275 xenografts or UOK342 xenografts were treated with dasatinib by oral gavage (25 mg/kg/week days) or vehicle . Tumors were measured on a weekly basis and, after six to seven weeks, tumors were excised and the mice sacrificed. As shown in Figure NF2-deficient PRCC tumors that have lost regulation by the Hippo signaling pathway.Next, we evaluated the NF2, a key regulator of the Hippo signaling pathway was observed in 3.6% of PRCC and, in combination with copy number loss, NF2 alterations were present in 22.5% PRCC, including both type 1 and type 2 PRCC histologies [Papillary renal cell carcinoma (PRCC) is a genetically and histologically heterogeneous disease. The Cancer Genome Atlas Research Network's recent comprehensive analysis of the molecular and genetic features of PRCC has greatly improved our understanding of the pathways involved in PRCC carcinogenesis and is opening novel targeted therapeutic opportunities. Mutation of NF2 mutations; one derived from a patient with type 2 PRCC, UOK275, and one derived from a patient with type 1 PRCC, UOK342. In addition, a known, commonly used type 1 PRCC cell line, ACHN, had been reported to have a NF2 mutation and this was confirmed within this study. A series of siRNA experiments demonstrated that all 3 NF2-deficient PRCC cell lines were dependent on YAP1 transcriptional activity to support their survival and growth. Similar to that observed within β-catenin active tumors [NF2-deficient PRCC cell lines and for YAP1 transcriptional activity. YAP1 transcriptional activity is regulated at the posttranslational level by phosphorylation by multiple kinases, including both the downstream members of the Hippo signaling pathway and several members of the src kinase family, including Yes [NF2-deficient PRCC cell lines could be specifically targeted with dasatinib and saracatinib in a predominantly Yes-dependent manner. These data were further complemented by showing that Yes kinase inhibitors induced a G0/G1 arrest in all 3 NF2-deficient PRCC cell lines, but had no effect on the HEK293 control cell lines, while the alternative src inhibitors had no effect on cell cycle in any cell line.We identified 2 PRCC cell lines bearing e tumors , Yes actding Yes –27. DasaNF2-deficient PRCC, the disruption of the Hippo pathway and aberrant activation of YAP1 are likely to promote cell proliferation. The YAP1 downstream targets cyclin D1 (CCDN1) and survivin (BIRC5) could both regulate the cell cycle. Cyclin D1 expression is required and maintained during the G1 phase and for the initiation of S phase, after which it is degraded. Survivin is part of the inhibitor of apoptosis (IAP) family and is only expressed in G2/M phase. It is thought that survivin affects the M phase by binding to mitotic spindles. The G0/G1 arrest observed in the treated cell lines is most likely due, at least in part, to the decrease in cyclin D1 expression.The Hippo pathway controls organ size and cell proliferation by regulation of YAP1 cellular localization and transcriptional activity. In in vitro analysis had demonstrated the effectiveness of Yes inhibitors on cell lines, so we proceeded to assess the effect of dasatinib in vivo on the growth of two NF2-deficient PRCC xenograft models using the type 2 PRCC cell line UOK275 and the type 1 PRCC cell line UOK342. Both models presented a tumor growth inhibition in the dasatinib-treated groups compared with the vehicle-treated groups. Taken together our data identify both Yes kinase activity and YAP1 transcriptional activity as attractive therapeutic targets in NF2-deficient PRCC.The NF2 mutation has paved the way for the development of novel targeted therapies. This preclinical study is providing some scientific rationale as to why to target Yes kinase activity and identifies Yes inhibitors (such as dasatinib) as promising potential therapeutic agents for patients with NF2-deficient PRCC tumors.The recent uncovering of a subgroup of PRCC patients with UOK275 and UOK342 cell lines were established in the Urologic Oncology Branch from tumor specimens . A full characterization of the cell lines will be published elsewhere. Briefly, UOK275 was established from a 10 cm retroperitoneal mass that proved to be metastatic RCC in a 55-year-old male who had undergone a right radical nephrectomy the year before for multifocal renal masses involving a 9 cm area of the right kidney and invading the renal vein. UOK342 was established from ascites fluid taken from a 63-year-old male with a history of type 1 PRCC. The immortalized human embryonal kidney cell line HEK293 and the papillary kidney tumor cell line ACHN were both purchased from ATCC . Cells were cultured in high glucose DMEM containing pyruvate and glutamine and supplemented with 10%FBS. The cells were harvested or treated when they reached 70–80% confluence.NF2 were validated by PCR using a Qiagen Taq PCR Core Kit  per the manufacturer's specifications, followed by bidirectional sequencing using the BigDye Terminator v.1.1 Cycle Sequencing Kit  per the manufacturer's specifications. Sequencing was performed on an ABI 3730/ABI 3130xl Genetic Analyzer automated sequencing machine . Sanger Sequencing was conducted at the CCR Genomics Core at the National Cancer Institute. Forward and reverse sequences were evaluated using Sequencher 5.4.6 . NF2-specific primers used were exon 2F: GAGAGTGGAGAGTGCAGAGA, exon 2R: CTTCTCTACTATACAGCTACAG, exon 9F: GGTTTAGTGCCTGGATACTG, exon 9R: CTTCACAAGATGTCACTCTGA, exon 12F: ACAGCACATGATCCCACTTC, exon 12R: GACAACTGCTGTAGAGCTCA.DNA was extracted from cell pellets using a Promega Maxwell 16 Cell DNA Purification Kit . UOK275 and UOK342 cell line DNA was assessed using the OncoVar assay as previously described , 29. MutDasatinib, sarcatinib, PP2 and WH-4-023 were purchased from Selleck Chemicals . Smartpool ON-TARGETplus siRNAs targeting YAP and YES were purchased from GE Healthcare .Ten to twenty micrograms of protein were loaded in 4–20% polyacrylamide gels . After electrophoresis, proteins were transferred to PVDF membranes, blocked with 5% fat-free milk for 1h, and incubated with primary antibodies overnight at 4°C (dilution 1:1000). After 3 × 10 min washes with TBS-Tween, blots were incubated with horseradish peroxidase-linked secondary antibodies  for 2 h before development with the ECL protein detection system . Rabbit antibodies against survivin (#2808), YAP (#4912), and cyclin D1 (#2922), and mouse antibodies against β-actin (#3700) and α-tubulin (#3873) were from Cell Signaling Technology, Inc . Rabbit antibody against CTGF (#ab5097) and phospho-YAP (Tyr357) (#ab62751) was from Abcam .Cell viability was measured using a Cell-Titer Glo purchased from Promega Biosciences, Inc.  following the manufacturer's protocol and as previously described .® VILO™ cDNA Synthesis Kit  in a 20 μL volume and diluted 10-fold with RNase-free water for downstream applications. RT-PCR amplification was performed using an ABI ViiA7 real-time PCR system  and TaqMan® Gene Expression Assays  using 2 μL of dilute cDNA for each reaction. All reactions were performed in triplicate using the standard ViiA7 reaction template and expression was normalized to the 18S control gene (Hs99999901_m1) and calculated using the ViiA7 software as comparative CT (ΔΔCT) values. The following set probes were used: BIRC5 (Hs03043574_m1), CTGF (Hs01026927_g1), and CCND1 (Hs00765553_m1).Cell lines were grown to approximately 80–90% confluency, washed with 5 mL of sterile PBS, and RNA was extracted using the RNeasy mini kit  in accordance with the manufacturer's protocol. RNA was eluted in 50 μL of RNase-free water and the measured using a NanoDrop 2000 UV-Vis Spectrophotometer . Complementary DNA was generated from 2 μg of RNA using the SuperScriptTwo thousand cells were seeded in 96-black well plates (Corning). The following day, cells were fixed with 4% paraformaldehyde for 1 hour and permeabilized with 1% triton for 10 minutes. After blocking with 1% bovine serum albumin (BSA)/PBS for 1 hour, the cells were incubated overnight with FITC-conjugated rabbit YAP antibody  in 0.1% BSA/PBS at 4°C. After being washed with PBS, cells were incubated 10 mg/mL Hoechst 33342 and Texas Red phalloidin (Invitrogen) in 0.1% BSA/PBS for 1 hour. Cells were washed 3 times with PBS prior to image acquisition. Image acquisition for each well was performed on an IN Cell Analyzer 2000  by using a 10x objective lens. YAP nuclear/cytoplasmic localization was determined with the IN Cell Developer software  by using Hoechst staining to define nuclear regions and phalloidin staining to define cells cytoplasm.Cells were incubated with 10 μM BrdU for 2 hours prior harvest. Cells were then trypsinized and fixed overnight at 4°C in 70% ethanol in calcium- and magnesium-free phosphate buffered saline (PBS). Ethanol solution was removed and cells were incubated in 3 mL of 0.08% pepsin in 0.1 N HCl at 37°C for 20 minutes. Pepsin was removed and nuclei were incubated in 1.5 mL of 2 N HCl at 37°C for 20 minutes. The nuclei containing acid solution was neutralized with 3 mL of 0.1 M sodium borate. Nuclei were spun out of neutralized acid and washed with 2 mL IFA buffer  and then incubated overnight at 4°C with anti-BrdU clone MoBU-1 conjugated to AlexaFluor488 (Invitrogen B35130) in IFA buffer. DNA was stained for 30 minutes with IFA buffer with of 50 μg/mL propidium iodide (PI) (Sigma-Aldrich #P4864) and 5 μg/mL RNase A (Sigma #R4642). Cell cycle analysis (bivariate plots of BrdU incorporation and DNA content) was performed on a FACSCanto II (Becton Dickinson). Data were collected and analyzed using FlowJo software .3. Mice were randomized into four groups of ten mice bearing either UOK342 or UOK275. One group of UOK342 and one group of UOK275 were treated with dasatinib  while the other groups received the vehicle (water/PEG 1:1) in a similar manner. After 6-7 weeks treatment, animals were sacrificed.Animal experiments were performed in accordance with the guidelines of the Animal Care and Use Committee of the National Institutes of Health. Forty female athymic nude mice  were injected on the right flank with 2 million of UOK342 or UOK275 cells diluted in matrigel ; 20 mice per cell lines). Two weeks after injection, tumor volume reached > 100 mmP < 0.05 was considered significant.All values are expressed as mean ± standard error. All experiments have been performed three times, with exception of the animal study. Values were compared using the Student-Newman-Keul's test.


Cysteine dioxygenase 1 is a metabolic liability for non-small cell lung cancer. Published 20, May 2019Figure 3, Figure 3—figure supplement 2B, Figure 5—figure supplement 3A and Figure 5—figure supplement 4C have an erroneous label that groups the PC9 cell line with KEAP1 mutant cell lines. This cell line, while previously characterized as having high NRF2 activity :1475-81), do not have a KEAP1 mutation. This error does not affect our conclusions, but may lead to confusion in the field about the mutation status of this cell line, which we would like to avoid. Thus, this labelling error is being changed as following.LOW and NRF2HIGH NRF2 NSCLC cell lines expressing CDO1Y157F. (Bottom) Intracellular cysteine concentration of the parental cell lines (minus doxycycline). (Right) Correlation between CDO1 protein levels and intracellular cysteine concentrations. CDO1 protein was normalized to α-Tubulin. N = 13.(d) (Top) Western blot analysis of NRF2, CDO1 and α-Tubulin expression in KEAP1 wild-type and KEAP1 mutant NSCLC cell lines expressing CDO1Y157F. (Bottom) Intracellular cysteine concentration of the parental cell lines (minus doxycycline). (Right) Correlation between CDO1 protein levels and intracellular cysteine concentrations. CDO1 protein was normalized to α-Tubulin. N = 13.(d) (Top) Western blot analysis of NRF2, CDO1 and α-Tubulin expression in LOW and NRF2HIGHNRF2 NSCLC cell lines. Mouse liver is included as a positive control for GOT1 and CSAD expression. Cells were fed with fresh RPMI +10% FBS 4 hr prior to lysate collection.(a) Western blot analysis of GOT1, CSAD and HSP90 levels in KEAP1 wild-type and KEAP1 mutant NSCLC cell lines. Mouse liver is included as a positive control for GOT1 and CSAD expression. Cells were fed with fresh RPMI +10% FBS 4 hr prior to lysate collection.(a) Western blot analysis of GOT1, CSAD and HSP90 levels in LOWNRF2 (gray) and HIGHNRF2 (red) NSCLC cell lines of cystine for 3 days. Cell numbers were analyzed with crystal violet and normalized to cells grown in cystine-containing media. N = 3 replicates/group.(b) Analysis of cell viability following starvation of KEAP1 wild-type (gray) and KEAP1 mutant (red) NSCLC cell lines of cystine for 3 days. Cell numbers were analyzed with crystal violet and normalized to cells grown in cystine-containing media. N = 3 replicates/group.(b) Analysis of cell viability following starvation of LOWNRF2 (gray) and HIGHNRF2 (red) NSCLC cell lines with 0–10 mM Na2SO3 for 3 days. Viable cells were analyzed with CellTiter-Glo and normalized to untreated cells. N = 3 replicates/group.(c) Analysis of cell viability following treatment of KEAP1 wild-type (gray) and KEAP1 mutant (red) NSCLC cell lines with 0–10 mM Na2SO3 for 3 days. Viable cells were analyzed with CellTiter-Glo and normalized to untreated cells. N = 3 replicates/group.(c) Analysis of cell viability following treatment of WT or a catalytically inactive CDO1 mutant  at single copy into the panel of LOW and NRF2HIGH NSCLC cell linesNRF2 . The level of CDO1 protein expression in these cells was similar with the physiological Cdo1 levels in mouse lung and liver , with liver being one of the highest CDO1-expressing tissues that is responsible for supplying TAU to the body . We find that CDO1 accumulated to higher levels in HIGH cells than NRF2LOWNRF2, although accumulation was observed in many LOWNRF2 cell lines as well .To investigate the NRF2-dependent regulation of CDO1 protein in NSCLC, we generated a doxycycline-inducible lentiviral expression system to reintroduce GFP, CDO1WT or a catalytically inactive CDO1 mutant  at single copy into the panel of WT and KEAP1MUT NSCLC cell linesKEAP1 . The level of CDO1 protein expression in these cells was similar with the physiological Cdo1 levels in mouse lung and liver , with liver being one of the highest CDO1-expressing tissues that is responsible for supplying TAU to the body . We find that CDO1 accumulated to higher levels in MUT cells than KEAP1WTKEAP1, although accumulation was observed in many WTKEAP1 cell lines as well .To investigate the NRF2-dependent regulation of CDO1 protein in NSCLC, we generated a doxycycline-inducible lentiviral expression system to reintroduce GFP, CDO12SO3, but not HTAU, led to cytotoxicity . We found that (CYS)2 starvation and Na2SO3 treatment were universally toxic to NSCLC cell lines, which did not depend on NRF2 activity . In addition, CDO1, CSA and Na2SO3 sensitized A549 cells to oxidative stress , consistent with their ability to deplete (CYS)2. Collectively, these results demonstrate that CSA and SO32- are toxic to NSCLC cells regardless of NRF2 activity, suggesting that resistance to (CYS)2 starvation is not an inherent phenotype of HIGH cellsNRF2. Rather, they are sensitive to CDO1 expression due to high intracellular CYS and CDO1 stabilization.Next, we examined the toxicity of CDO1 products to NSCLC cell lines. Treatment of cells with CSA and Na2SO3, but not HTAU, led to cytotoxicity . We found that (CYS)2 starvation and Na2SO3 treatment were universally toxic to NSCLC cell lines, which did not depend on KEAP1 mutation status . In addition, CDO1, CSA and Na2SO3 sensitized A549 cells to oxidative stress , consistent with their ability to deplete (CYS)2. Collectively, these results demonstrate that CSA and SO32- are toxic to NSCLC cells regardless of KEAP1 mutation status, suggesting that resistance to (CYS)2 starvation is not an inherent phenotype of KEAP1 mutant cells. Rather, they are sensitive to CDO1 expression due to high intracellular CYS and CDO1 stabilization.Next, we examined the toxicity of CDO1 products to NSCLC cell lines. Treatment of cells with CSA and NaThe article has been corrected accordingly.


RAS wild-type CRC cell lines with different cetuximab sensitivities. CRC cell lines with different sensitivities to cetuximab showed rather low EGFR expression but high HER2 and HER3 expression. These results were in line with the The Cancer Genome Atlas (TCGA) data from CRC patients, where higher mRNA levels of HER2 and HER3 were also detected compared to EGFR. Therefore, the targets of afatinib were indeed expressed on the CRC cell lines used in this study and in CRC patients. Furthermore, cetuximab resistance had no significant influence on the expression levels of HER receptors in CRC cell lines (p ≥ 0.652). This study also demonstrated that afatinib was able to induce a concentration-dependent cytotoxic effect in RAS wild-type CRC cell lines with different cetuximab sensitivities. Neither cetuximab resistance (p = 0.233) nor hypoxia (p = 0.157) significantly influenced afatinib’s cytotoxic effect. In conclusion, our preclinical data support the hypothesis that treatment with afatinib might be a promising novel therapeutic strategy for CRC patients experiencing intrinsic and acquired cetuximab resistance. The epidermal growth factor receptor (EGFR) is an important therapeutic target in colorectal cancer (CRC). After the initial promising results of EGFR-targeted therapies such as cetuximab, therapeutic resistance poses a challenging problem and limits the success of effective anti-EGFR cancer therapies in the clinic. In order to overcome resistance to these EGFR-targeted therapies, new treatment options are necessary. The objective of this study was to investigate the expression of human epidermal growth factor (HER) receptors and the efficacy of afatinib, a second-generation irreversible EGFR-tyrosine kinase inhibitor, in  RAS wild-type metastatic CRC (mCRC) when given in combination with FOLFIRI  and irinotecan) and with FOLFOX , respectively [RAS patients [RAS wild-type mCRC, the addition of cetuximab to FOLFIRI and panitumumab to FOLFOX resulted in a median overall survival of 23.5 months and 25.8 months versus 19.5 months and 20.2 months with chemotherapy alone, respectively [RAS wild-type disease, 40–60% of patients fail to respond, possibly due to mechanisms that can compensate for reduced EGFR signaling or mechanisms that can modulate EGFR-dependent signaling [Increased or sustained signaling of the epidermal growth factor receptor (EGFR) plays an integral role in the tumorigenesis of many cancer types, including colorectal cancer (CRC), making it a compelling drug target. Inhibition of EGFR signaling has been a focus of research over the last decades and this has led to the development of multiple EGFR-targeted agents. The EGFR-targeted monoclonal antibodies (mAbs) cetuximab and panitumumab have already demonstrated a significant survival improvement in patients with ectively ,4,5,6,7.patients . In RAS ectively . Neverthignaling ,15,16,17The precise mechanisms of intrinsic and acquired resistance to EGFR inhibitors remain unclear. Since EGFR signaling is prominent in CRC, the inhibition of this EGFR pathway is still considered as an important therapeutic strategy. Extensive dimerization among the different human epidermal growth factor (HER) receptor tyrosine kinases suggests that blocking signaling from more than one family member may be essential to effectively treat CRC and limit drug resistance . In contOur previous preclinical research showed not only that afatinib displays a cytotoxic effect in CRC, but also demonstrates effective cytotoxic activity in intrinsic and acquired cetuximab-resistant head and neck squamous cell carcinoma (HNSCC) cell lines ,24. HoweRAS wild-type CRC cell lines that are either sensitive or have intrinsic/acquired resistance to cetuximab. With this in mind, we decided to: (1) examine the expression of HER receptors in CRC in order to determine the presence of afatinib’s targets, (2) test the influence of cetuximab resistance on the expression of HER receptors in RAS wild-type CRC cell lines, (3) determine the cytotoxic effect of afatinib in these RAS wild-type CRC cell lines with different cetuximab sensitivities, (4) study the efficacy of afatinib under both normal and reduced oxygen conditions, as CRC is often characterized by regions with reduced oxygen levels and as there is a link between hypoxia and EGFR signaling [This study aims to provide preclinical data concerning the expression of HER receptors and the potential of afatinib in a panel of ignaling , (5) exaRAS wild-type CRC cell lines (50) values, two out of four CRC cell lines  were considered as intrinsically resistant to cetuximab, as the percentage of viable cells in these cell lines did not decrease below 50%. CaCo2 and Lim1215 were identified as cetuximab-sensitive, as the IC50 values  are considered as clinically relevant [Sensitivity to cetuximab therapy was investigated in a panel of ll lines A. Based relevant . Next, cAs afatinib binds to multiple members of the HER receptor family, the basal cellular membrane protein expression level of these HER family members was determined in our panel of CRC cell lines with different sensitivities to cetuximab. p ≥ 0.652) and ΔMFI of receptor-positive cells (p ≥ 0.695) were observed between cetuximab-sensitive, intrinsically-resistant, and acquired cetuximab-resistant CRC cell lines. HER4 expression was barely observed in any of the CRC cell lines tested and when detected, HER4 expression levels were very low (data not shown).EGFR expression was observed in all the CRC cell lines used in this study. However, the percentage of EGFR-positive cells ranged between 11% and 34% A. Moreovp < 0.001 and p < 0.001, respectively). This corresponds with our flow cytometric findings in CRC cell lines. Moreover, CRC patients also demonstrated very low HER4 mRNA expression, which is in line with our flow cytometric data in CRC cell lines (data not shown). Next, RNA sequencing data from the The Cancer Genome Atlas (TCGA) dataset of CRC patients  was used to compare our in vitro results ,28. The Overall, these results demonstrate that CRC cell lines with different sensitivities to cetuximab show rather low EGFR expression but high HER2 and HER3 expression. These results were in line with the TCGA data from CRC patients, as also in patient material, whereby higher mRNA levels of HER2 and HER3 were detected compared to EGFR. Furthermore, cetuximab resistance had no influence on the expression levels of HER receptors in CRC cell lines. We therefore concluded that the targets of afatinib were indeed expressed on the CRC cell lines used in this study and in CRC patients.50 values for afatinib under normoxia ranged from 0.007 ± 0.002 μM to 2.379 ± 0.869 μM . Furthermore, no statistical difference in the cytotoxic effect of afatinib was observed when cells were treated under hypoxic conditions (p = 0.157).The cytotoxic effect of the irreversible HER family blocker afatinib was studied in cetuximab-sensitive, intrinsically/acquired-resistant, and PBS-treated control CRC cell lines under both normoxic and hypoxic conditions. A clear concentration-dependent cytotoxic effect of afatinib (0–10 μM) after 72 hours of treatment was observed in all CRC cell lines . The IC50.869 μM . IntrinsOverall, afatinib showed a clear concentration-dependent cytotoxic effect in cetuximab-sensitive and intrinsically-resistant CRC cell lines and CRC cell lines with acquired resistance. Our results demonstrated that neither cetuximab resistance nor exposure to hypoxia provoked therapeutic resistance to afatinib.0/G1 cells  and a decrease in the percentage of S phase cells . A significant decrease in the percentage of S cells was also found in CaCo2-R (p ≤ 0.033). In contrast, a significant increase in the percentage of G2/M cells was observed in CaCo2 (p ≤ 0.028), CaCo2-PBS (p = 0.039) and Lim1215-R (p = 0.047) after treatment with high dose of afatinib (IC60 and IC80). Overall, afatinib did not have a pronounced effect on the cell cycle distribution of CRC cells, independently of their sensitivity to cetuximab. We studied the effect of afatinib on the distribution of CRC cells in the different phases of the cell cycle was investigated . SW48 an80 /propidium iodide (PI) staining . This te80 .20 and IC40 values specific for each cell line. The dose–response curves after treatment with both sequential combination regimens are shown in 50 values ranging from 0.09 ± 0.02 μM to 40.07 ± 4.59 μM, not including CaCo2-PBS and CaCo2-R , except in the HT29 cell line (p = 0.023). Furthermore, the combination index (CI) ranged from 0.81 ± 0.10 to 1.12 ± 0.11, indicating an additive interaction. Similarly, treatment with irinotecan followed by afatinib showed no significant decrease in IC50 value (p ≥ 0.116) and CI ranged from 0.72 ± 0.13 to 1.22 ± 0.32, revealing additive to subadditive effects. Thus, sequential exposure to afatinib followed by irinotecan or the reverse regimen  revealed additive, yet no synergistic interactions.In order to investigate the potential interaction between afatinib and irinotecan, CRC cells were incubated with fixed doses of afatinib for 72 hours, followed or preceded by treatment with 0–50 μM irinotecan for 24 h. The fixed afatinib concentrations were based on the outcome of the monotherapy experiments and corresponded to the IC CaCo2-R . ConcernRAS wild-type CRC cell lines was determined. Afterwards, the cytotoxicity of afatinib, as a single agent and in combination with irinotecan, was examined in this panel of RAS wild-type CRC cell lines that are either sensitive or have intrinsic/acquired resistance to cetuximab.Targeted therapies are the key for the personalized treatment of cancer patients. After initial promising results of EGFR-targeted therapies such as cetuximab, therapeutic resistance poses a challenging problem and limits the success of effective anti-EGFR cancer therapies in the clinic. As a result, new treatment options are needed to overcome drug resistance. Due to the intensive interactions between HER receptors, inhibition of one HER receptor can be compensated by other HER family members, which therefore must be targeted by new therapeutic regimens. In contrast to the first-generation EGFR-inhibitors, afatinib irreversibly blocks EGFR, HER2, and HER4. Consequently, we hypothesized that treatment with afatinib might result in a more pronounced therapeutic benefit, even in patients who experience resistance to first-generation EGFR inhibitors. To investigate this hypothesis, we first examined the expression of HER receptors in CRC in order to determine the presence of afatinib’s targets. In addition, the influence of cetuximab resistance on the expression of HER receptors in RAS wild-type CRC cell lines with different cetuximab sensitivities and compared these results with RNA sequencing data from the TCGA dataset of CRC patients. According to the target expression of EGFR and HER2, the CRC cell lines used in this study are valid candidates for treatment with afatinib. These in vitro results were in line with the TCGA data from CRC patients, who also express EGFR and HER2, indicating the presence of afatinib’s targets in CRC. Interestingly, both CRC cell lines and patients demonstrated higher HER2 and HER3 expression compared to EGFR expression. It has already been suggested that HER2 expression might play an important role in response to afatinib treatment in CRC [In the first part of this study, we determined the expression of HER receptors in t in CRC ,30. Conft in CRC ,32, resut in CRC . In addit in CRC ,35,36,37Resistance to cetuximab has been associated with extensive dimerization among the different HER receptor tyrosine kinases . In the KRAS wild-type CRC patients was predictive for efficacy of panitumumab [Several studies have demonstrated a correlation between HER2 amplification and acquired resistance to cetuximab in CRC ,40. Furtitumumab . Althougitumumab . Neverth50 values of SW48, HT29, and CaCo2-R cell lines are higher than this maximum afatinib plasma concentration. In the future, it would be highly interesting to further investigate the efficacy of afatinib in long-term in vitro assays. For example, in the past, Herr et al. have already determined the long-term effect of afatinib treatment on the HT29 CRC cell line in a clonogenic assay  [In the second part of this study, we demonstrated that afatinib has the potential to overcome intrinsic and acquired cetuximab resistance, as it was able to establish a cytotoxic effect in CRC cell lines with different sensitivities to cetuximab. Camidge et al. have recently demonstrated in a phase Ib trial that the maximum plasma concentration of afatinib at its maximum tolerated dose was 313 ng/mL, which corresponds to 0.644 μM [14 days) . They re0/G1 cell cycle arrest and induced apoptotic cell death in different cancer cell lines, including CRC cell lines [0/G1 cell cycle arrest or induction of apoptosis. Therefore, analyzing caspase activity and markers of autophagy and other types of cell death after afatinib treatment, would be highly interesting to study more in depth. Thus, additional research is necessary to further unravel the mode of action of afatinib in CRC. Importantly, exposure to hypoxia did not provoke therapeutic resistance to afatinib in CRC cell lines. This is an interesting finding as oxygen deficiency is a common characteristic of CRC and these hypoxic tumor regions often contain viable cells that are more resistant to conventional therapies [Concerning the molecular mechanisms underlying afatinib’s cytotoxic effect in CRC, previous studies demonstrated that afatinib caused a Gll lines ,46,47,48herapies .50 values compared to cetuximab-sensitive CRC cell lines, thus suggesting the possibility of cross-resistance between cetuximab and afatinib. Previous studies in HNSCC have indicated that afatinib is more effective in patients whose tumors are cetuximab-naïve [Despite the fact that statistical analysis did not reveal a significant effect of cetuximab resistance on the cytotoxicity of afatinib, we noticed that intrinsically and acquired cetuximab-resistant CRC cell lines tended to show higher ICab-naïve ,50,51. CKRAS wild-type CRC patients [KRAS-mutated CRC cell lines, no clinical benefit of afatinib was observed in KRAS mutated patients in this phase II study. Previous treatment history might have had an impact on the lack of clinical benefit observed in the study. Recently, interest in afatinib as monotherapy for the treatment of CRC is diminished, indicated by the low number of novel clinical trials in CRC patients found on ClinicalTrials.gov. However, afatinib did demonstrate promising clinical efficacy in other solid tumors, including HNSCC and non-small-cell lung carcinoma (NSCLC) [In the past few years, afatinib has been investigated in various clinical trials. In phase I studies, some promising signs of antitumor activity after afatinib treatment were observed in patients with advanced solid tumors, including CRC. After these promising phase I dose-escalation studies, afatinib was further investigated in several phase II studies. Hickish et al. reported that afatinib (40 mg/day) demonstrated inferior response and survival compared to cetuximab in patients . Despite (NSCLC) ,53,54.As most adjuvant cancer treatments are combinations of chemotherapeutic agents and/or radiotherapy, we are convinced that new EGFR-targeted agents, such as afatinib, will achieve their greatest efficacy in combination with traditional cytotoxic agents and/or radiotherapy. As cetuximab has been approved for the treatment of mCRC in combination with the irinotecan containing chemo-regimen FOLFIRI, we investigated the combination of afatinib with irinotecan in CRC cell lines that are sensitive and have intrinsic/acquired resistance to cetuximab. In clinical studies, it has already been reported that simultaneous treatment with afatinib and standard chemotherapy is associated with increased frequency of side effects ,56. TherBesides combining afatinib with standard chemotherapy and/or radiotherapy, combinations with other targeted agents such as vascular endothelial growth factor inhibitors have been investigated in clinical studies, with limited success in CRC patients ,58. In aAs mentioned above, interest in afatinib for the treatment of CRC has diminished, and currently no novel studies have been initiated to investigate combinations of afatinib with other drugs for the treatment of CRC patients . However, as our and other preclinical studies have demonstrated the potential of afatinib in CRC cell lines, its limited clinical benefit in CRC patients could be explained by the lack of predictive biomarkers necessary for optimal patient selection. We foresee that liquid biopsies can play a key role in the progress needed for improving the efficacy of targeted therapies in CRC. Liquid biopsies will help to identify and monitor the biomarkers for both response and resistance to EGFR-targeted therapies in CRC as well as speed up the process of patient selection . As suchRAS wild-type. SW48, HT29, and CaCo2 were cultured in DMEM medium, while Lim1215 was grown in RPMI medium . Each medium was supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% L-glutamine . Sodium pyruvate was additionally added to RPMI medium. Cells were grown as monolayers and maintained in exponential growth in 5% CO2/95% air in a humidified incubator at 37 °C. Authenticity of cell lines was verified by establishing short tandem repeat profile. All cell lines were confirmed free of mycoplasma infection through regular testing .The human CRC cell lines SW48, HT29, and CaCo2 were purchased from the American Type Culture Collection . Lim1215 was obtained from CellBank Australia . All cell lines were Afatinib for 72 h immediately followed by irinotecan for 24 h;Irinotecan for 24 h immediately followed by afatinib for 72 h.Cell survival was determined using the colorimetric sulforhodamine B (SRB) assay, as previously described ,61. This50 values  were calculated using WinNonlin software . Possible synergism between afatinib and irinotecan was determined by calculation of the combination index (CI) using the ‘Additive Model’ as described by others [The pharmaceuticals, cetuximab  and irinotecan  were diluted in sterile PBS. Afatinib  was diluted in DMSO  and further dilutions were made in cell culture medium. Survival rates were calculated by:  of treated cells/mean OD of control cells) × 100%. ICy others ,63,64. C2) was achieved in a Bactron IV anaerobic chamber , as described previously [2.Hypoxia . After 10 dose doublings, dose–response studies were determined for each resistant cell line (suffix R). In order to examine whether acquired resistance was a transient or permanent effect, dose-response curves of cetuximab were re-assessed in the resistant cell lines after 6 weeks in culture without cetuximab.Generation of cell clones with acquired resistance was performed as described previously ,66,67. A6 cells, 25μg antibody in 1ml, R&D Systems, Minneapolis, MN, USA). Corresponding isotype controls  were included for all samples and served as negative controls. Dead cells were excluded from the analysis by staining with 7-AAD . All samples were measured on a FACScan flow cytometer (BD Biosciences). Flow cytometric data were analyzed using FlowJo v10.1 . The percentages of EGFR, HER2, HER3, and HER4-positive cells (Overton) were determined in comparison with the corresponding isotype control using the Overton method. Furthermore, the signal for aspecific binding was subtracted from the MFI (=ΔMFI). This parameter indicates the amount of extracellular expression of EGFR, HER2, HER3, and HER4 on individual cells. The baseline extracellular protein expression level of EGFR, HER2, HER3, and HER4 was assessed using flow cytometry, as previously described ,67. CellThe obtained results were compared using the RNA sequencing data from the TCGA dataset of CRC patients . RNASeqV2 from TCGA was processed and normalized using the software package RSEM (RNA-Seq by Expectation Maximization) to generate transcripts per million. This dataset was downloaded from cBioportal.20, IC40, IC60, and IC80 values specific for each cell line. Cell cycle distribution was determined using the CycleTESTTM PLUS DNA reagent kit (BD Biosciences). Induction of apoptotic cell death was investigated flow cytometrically using the AnnV-FITC/PI assay (BD Biosciences). Both assays were performed on a FACScan flow cytometer and analyzed with FlowJo v10.1. After overnight incubation, cells were treated for 72 h with afatinib. Since the sensitivity to afatinib strongly varied between the cell lines, afatinib concentrations were based on the outcome of the monotherapy experiments and corresponded with the ICAll experiments were performed at least three times independently, unless otherwise stated. In cytotoxicity experiments, each condition was tested in triplicate in each of the three independent experiments. Flow cytometry experiments were independently performed three times with one sample for each condition. Results are presented as mean  ± standard deviation. The effects of various conditions and treatments were studied using linear regression or linear mixed models in case of non-independent observations. All models were fitted using a stepwise backward strategy, starting from a model with all fixed effects and their interactions. If the interaction term was not significant, a model with only the main effects was fitted. If the treatment effect was significant, a Dunnet posthoc analysis was performed. The effect of cetuximab resistance on the expression of HER receptors was analyzed using a linear mixed model with cetuximab resistance status as fixed effect. A random intercept for cell line was added, in order to account for the dependence between observations within the same cell line. Significant differences in HER receptor mRNA expression of CRC patients (RNASeqV2 TCGA data) were assessed using one-way ANOVA with Bonferroni posthoc analysis.50 values in the combination experiments were investigated using one-way ANOVA with Dunnet posthoc analysis.Effects of oxygen and resistance status on afatinib’s cytotoxic effect were modeled using a linear mixed model with oxygen status, resistance status, and their interaction as fixed effects. A random intercept for cell line was added to account for the dependence between observations within the same cell line. Significant differences in the cell cycle distribution, induction of apoptotic cell death and ICp-values below 0.050 were considered significant.GraphPad Prism 7 was used for data comparison and artwork. All statistical analyses were performed in JMP Pro 13 and SPSS v24. RAS wild-type CRC cell lines with different sensitivities to cetuximab show rather low EGFR expression but high HER2 and HER3 expression. These results were in line with the TCGA data from CRC patients, where higher mRNA levels of HER2 and HER3 were also detected compared to EGFR. Consequently, the targets of afatinib are expressed on the CRC cell lines used in this study and in CRC patients. Cetuximab resistance had no influence on the expression levels of HER receptors in CRC cell lines. The present study also demonstrated that afatinib was able to establish a concentration-dependent cytotoxic effect in RAS wild-type CRC cell lines that are either sensitive or have intrinsic/acquired resistance to cetuximab. Neither cetuximab resistance nor hypoxia significantly influenced afatinib’s cytotoxic effect. Treatment with afatinib had little effect on the cell cycle distribution and the induction of apoptosis. Sequential combinations of afatinib with irinotecan demonstrated additive effects, yet no synergistic interactions. Although these preclinical data support the hypothesis that afatinib might be a promising novel therapeutic strategy for the treatment of RAS wild-type CRC patients experiencing cetuximab resistance, to date no survival benefit has been observed in clinical trials. Therefore, additional studies with biomarker-driven patient recruitment are required to further explore the efficacy of afatinib in CRC patients.This study demonstrated that