Prognostic markers for glioblastoma are lacking. Both intrinsic tumour characteristics and microenvironment could influence cancer prognostic. The aim of our study was to generate a pure glioblastoma cell lines and immune classification in order to decipher the respective role of glioblastoma cell and microenvironment on prognosis.n = 481 and Rembrandt, n = 180) for which clinical data, transcriptomic profiles and outcome were recorded. Transcriptomic profiles of 129 pure glioblastoma cell lines were clustered to generate a glioblastoma cell lines classification. Presence of subtypes of glioblastoma cell lines and immune cells was determined using deconvolution.We worked on two large cohorts of patients suffering from glioblastoma (TCGA, Glioblastoma cell lines classification defined three new molecular groups called oncogenic, metabolic and neuronal communication enriched. Neuronal communication-enriched tumours were associated with poor prognosis in both cohorts. Immune cell infiltrate was more frequent in mesenchymal classical classification subgroup and metabolic-enriched tumours. A combination of age, glioblastoma cell lines classification and immune classification could be used to determine patient’s outcome in both cohorts.Our study shows that glioblastoma-bearing patients can be classified based on their age, glioblastoma cell lines classification and immune classification. The combination of these information improves the capacity to address prognosis. The current treatment relies on surgical resection of gross tumour followed by radio-chemotherapy and adjuvant therapy with temozolomide. After such therapy, most patients experiment recurrence and only a few therapeutic options are available. Despite such therapies, median survival only reaches around 15 months.2 Proneuronal tumours seem to be associated with a better outcome, whereas mesenchymal tumours are related to a poorer survival.5 However, because GBM are composed of a complex microenvironment, it is difficult to determine if such classification corresponds to a classification of the tumour cell or of the microenvironment.To better understand this pathology and define subgroups of patients with particular molecular biology and particular prognosis or response to therapy, the Cancer Genome Atlas Consortium (TCGA) performed high-dimensional profiling and molecular classification of large series of GBM tumours. Unsupervised transcriptome analysis revealed four clusters, referred to as classical, mesenchymal, neural and proneural, which were tightly associated with specific genomic abnormalities.6 GBM cells secrete numerous chemokines, cytokines and growth factors that promote infiltration of various cells: astrocytes, pericytes, endothelial cells, circulating progenitor cells and a range of immune cells such as microglia, peripheral macrophages, myeloid-derived suppressor cells (MDSC), leukocytes, CD4+ T cells and Treg into the tumour.10 Some previous work underlined that immune infiltrate could be associated with GBM prognosis. Although CD8 T cells seem to be linked with a better prognosis, some other immune components such as Th17 or myeloid cells are associated with a poorer prognosis.12 Recent computational methods as deconvolution were reported for predicting fractions of multiple cell types in gene expression profiles of admixtures.19 These methods could be used to estimate immune infiltrate in a tumour sample.The GBM microenvironment is a complex milieu that ultimately promotes tumour cell transcriptomic adaptability and disease progression.While previous classification used classical hierarchical clustering of transcriptome of bulk tumour sample, GBM contains both stromal and tumour cells. Our aim is to use pure transcriptomic data of GBM cells to isolate a tumour cells signature without the interference of transcriptomic information from the stroma microenvironment. In this article, we proposed a new classification of GBM tumours based on the estimation of GBM tumour cell heterogeneity. A new deconvolution process was proposed to estimate the quantity of immune cells present in GBM tumours. Combining this information, we were able to compute a score per patient that was associated with outcome in two independent cohorts.The first step of our analysis was to cluster transcriptome of GBM cell lines. This clustering step revealed three clusters. A signature of this three clusters was built to allow quantification of these clusters in patients’ tumours. Once the quantification was done, patients were clustered according to the quantity of cell lines clusters in their tumours. This process showed three clusters of patients. Each group were enriched in one of the three cell lines cluster built before. The results of this step were a new classification of patients’ tumour based on GBM cell lines. Once this classification is done, immune cells inside patients’ tumours were quantified using deconvolution. Finally, a multivariate Cox model was built combining the new classification and immune quantification . GSE12824 involves five GBM cell lines in duplicate from five different patients, GSE23806 involves cell lines or neurospheres or stem-like cells in duplicate in different stimulating conditions, GSE9171 involve cell lines from different patients (16 cell lines), GSE104291 involves six cell lines in triplicate from different patients and GSE8537 involves one cell line from a patient in four different conditions. All cell lines samples were obtained through Affymetrix H133plus2 platform. This dataset was used to create three groups of cell lines. We then estimated for each patient the quantity of each cell line group in their tumour.We downloaded GBM cell lines transcriptomes from different studies on GEO public repository22 Both clinical and gene expression profiles were downloaded with the TCGA2STAT R package.23 The data were already normalised using the RMA algorithm. We removed patients with a ‘G-CIMP' molecular subtype because they have a particular survival behaviour. We decided in both cohorts to exclude G-CIMP tumours because G-CIMP patients have better prognosis than other patients  of 22.7 months vs 10.5 months for other patients). This population is rare in the TCGA cohorts (39 patients among 520) and may introduce a bias in the analysis. In addition, it is known that such population is enriched in Isocitrate deshydrogenase (IDH) mutated glioma.22 All these arguments support that G-CIMP is probably a secondary GBM, which is raised from IDH mutated low-grade glioma and thus needed to be excluded because of its different biology. The number of patients after removal of these patients is 481 in this cohort. The second cohort (Rembrandt) was downloaded from GEO under the accession number ‘GSE68848'. Clinical data were downloaded from the GDOC website (https://gdoc.georgetown.edu/gdoc/). The Rembrandt study gathers patients with different types of brain tumour. We only kept the GBM type of tumours resulting in 180 patients for this cohort.24 Gene expression profiles were obtained through the H133Plus2 Affymetrix platform.We used two different cohorts for this study. The first one is the Affymetrix H133A TCGA GBM cohort.25 A batch effect was observed between the two GBM tumours datasets due to the different Affymetrix platforms. This unwanted effect was corrected using the SVA package.26 We worked on the set of genes common to the two platforms representing 51% of genes for the H133plus platform and 86% for the H133A.Raw data were normalised using the RMA method from the affy package.27 A t-test was performed for each gene testing if the average expression of the gene in one group is different compared with the other groups. Genes with a false discovery rate corrected p < 0.05 were considered as differentially expressed.A differential gene analysis was performed to find genes differentially expressed between the three groups of GBM cell lines. The differential expression analysis was performed with the limma R package.28 The most significant KEGG pathway found for each group was used to name it.Using clustering method, three types of GBM cell lines could be identified. A pathway analysis was performed on the genes differentially expressed between each of the three groups of cell lines generated. The list of differentially expressed genes for each group was uploaded on the EnrichR website.2 Cross-validation was performed on the TCGA cohort to assess the validity of the model; resulting in a well-classified rate of 90%. This model was then used to predict the molecular subtype of the Rembrandt’s tumour samples.Information about the molecular subtypes was available for the TCGA cohort but not for the Rembrandt’s one. To predict them for this latter cohort, we built a GBM molecular subtype classifier using the TCGA cohort as the training data. A Prediction Analysis for Microarrays classification model was estimated using the already published list of 840 genes that discriminate the four subtypes.29  or OS. Survival probabilities were estimated using the Kaplan–Meier method and survival curves were evaluated using the log-rank test. We censored the DFS times at 12 months and the OS times at 24 months for both cohorts.A multivariate Cox model was built with the cell line classification and the proportions of immune cells estimated. Interactions between the classification and the proportions of cells were considered in the model.32 we forced the cell line classification to be kept in the model.A LASSO survival model was performed using the glmnet R package,The resulting model was then used to estimate a linear predictor value for each patient.The multivariate Cox model combining GBM cell lines classification and immune cells quantities, estimated by deconvolution, was built as follows:Two risk groups of patients were built based on this linear predictor value, using the median value as the threshold. This model was built on the TCGA cohort and validated on the Rembrandt cohort. The validation was performed using the same coefficients on both cohorts and the same cut-offs.The last model built is composed of the linear predictor of the LASSO model and the clinical variable age. From this model, we built three groups of patients . The groups were determined using first and third quartile of the computer score with the linear predictor and the age of the patients.https://github.com/Klopfe/Glioblastoma).Version 3.3.3 of R was used for the statistical analysis. Figures were performed using GraphPad version 7.03. All data and R code can be downloaded on Github .We had the objective to generate a pure tumour cell-intrinsic transcriptomic signature of GBM. To do this, we made the hypothesis that a GBM is composed of different subgroups of GBM cell types. We downloaded 129 samples of GBM cell lines from public repository Table . Using h29 . The patients’ samples were taken from the TCGA GBM cohort (n = 481),22 used as discovery cohort, and the validation of our observations was made on the Rembrandt’s cohort (n = 180).24In order to quantify if GBM samples contained different proportions of these three tumour cell types, we used a deconvolution algorithm adapted from Cibersortn = 81), an oncogenic-enriched group (n = 202) and a neuronal communication-enriched group (n = 198), which are constituted of a mixture of neuronal, proneuronal and classical tumours with an enriched presence of neuronal tumours in oncogenic-enriched group  implying that this new classification is not related to the classical molecular classification.We then compared this GBM cell lines classification with the classical molecular classification. The new GBM cell lines classification and the classical classification were found independent  . In contrast, in Rembrandt cohort classical molecular classification is significantly (log-rank p = 0.01) associated with OS. However, in this cohort proneural tumours were not significantly associated with better OS , p = 0.11, in comparison with mesenchymal tumours)  Figure . Proneurrs) Fig. , thus sup = 0.16) and DFS log-rank p = 0.29) , p = 0.06, in comparison with oncogenic-enriched group) . Similarly to the TCGA cohort, the neural communication-enriched group is associated with poorer OS , p = 0.02 in comparison with oncogenic-enriched group)  Fig.  and  Fig. . However) Figure . In the up) Fig. .29 to obtain absolute estimations of immune cells infiltrated in tumours. Deconvolution process allowed quantification of immune cells inside GBM tumours. The estimations showed a very low infiltration in most of the tumours analysed since most of the tumours have <10% of immune cells.To estimate the different types of immune cells in GBM samples, we used a deconvolution algorithm adapted from CIBERSORTUsing transcriptomes of pure immune cells, we could estimate quantities of lymphoids cells , Th1, Th2, Th17, naive CD8, effector memory CD8, EMRA CD8 and central memory CD8) and myeloid cells  in each tumour samples from both cohorts. We observed in both cohorts that proneural and classical GBM have the weakest infiltrate of immune cells Figs. . In the Hierarchical clustering using the deconvolution estimations revealed five clusters in the TCGA cohort Fig. . In the These estimations showed differences in terms of immune infiltrations between the different molecular subgroups and between cell lines classification subgroups.We tested each immune cell as a continuous variable using univariate Cox proportional models, and only central memory CD8 T cells were associated with poorer OS in all patients belonging to TCGA cohort Fig. . In the p = 0.002, n = 479)   Fig. . The sam56) Fig. .Fig. 5A p = 0.0002, Table p = 0.0003).Finally, we wanted to check the ability of our composite score to bring new information when compared with classical clinical variables used to determine the prognosis. Clinical variables such as age, sex, 6-O-Méthylguanine-ADN méthyltransférase (MGMT) methylation and type of treatment were only available for TCGA cohort (no patients harbour IDH mutation). We built a multivariate Cox model including all these clinical variables and another one with the clinical variables and our composite score taken as continuous variable. Our composite score remains strongly significant in the model , 5.582, p Together our data underlined that a combination of the molecular signature and estimation of immune infiltrate quantities could be used to address patients’ outcome in glioblastoma patients.2 Most studies proposed that proneural tumours are associated with better outcome and could correspond to secondary GBM, which arise from low-grade tumours and mesenchymal tumours, are more common in older patients and are associated with a poor outcome. In our two cohorts, we could not confirm these observations. In the Rembrandt cohort, proneural tumours were associated with better outcome, whereas in the TCGA cohort it corresponded to the group with the worst prognosis. Similar observations were previously performed by other groups,33 thus suggesting that molecular classification have a minor role for stratification of patients in clinical practice.Although GBM tumours are morphologically identical, some patients have different clinical outcomes and different response to therapy. Gene expression profiling of GBM was used to identify several transcriptomal subgroups.34 proposed a new classification using RNA sequencing of single cells isolated from GBMs to select only specific genes expressed in GBM cells. While few information is available concerning the prognostic role of this classification, the authors observed intratumoural heterogeneity with the presence multiple subtype of GBM cells in most of tumour sample. Based on this observation, we used alternative strategies to perform both tumour-intrinsic molecular classification and to evaluate intratumoural heterogeneity. Using GBM cell lines, we defined particular clusters of GBM cells, which were enriched in genes of oncogenic pathway, metabolic pathway or neuronal communication pathway. Metabolic-enriched cluster was associated with mesenchymal tumours and oncogenic-enriched cluster was associated with neural tumours. This new classification was associated with prognosis, and in both TCGA and Rembrandt cohorts, neuronal communication-enriched tumours were associated with a poorer prognosis. Interestingly, this new classification did not perfectly mirror the classical molecular classification.A previous study from Wang et al.35 that a decrease in the level of CD8+ Tumor infiltrated lymphocytes (TILs) and a decrease in the CD8+/CD4+ ratio was observed in high-grade GBM patients in comparison with low grade. GBM have a weak CD8 immune infiltrate, which can probably not control tumour growth due to their relative low level compared with immunosuppressive cells. While a first report showed that a high CD8 T cells infiltrate was more frequently observed in longer survivors compared with shorter survivors,35 further studies suggested that CD8 alone could not be associated with clinical outcome.35 In addition, recent reports suggest that CD8 and CD4 T cells are exhausted in GBM thus suggesting their incapacity to drive an effector immune response.36 In a previous work, we observed in a series of GBM a good prognostic role of CD8 infiltrate and a deleterious role of Th17 cells; however, immune variables were not studied as continuous variables, which could induce some bias.11 Using CIBERSORT in the TCGA cohort, Wang et al.34 recently observed high proportions of macrophage, neutrophil and CD4 T cells in mesenchymal tumours. Our deconvolution strategy gives an additional information to CIBERSORT. CIBERSORT estimates the proportion of immune cells in the total number of immune cells that infiltrate the tumour and thus only gives an idea of the proportions of each cell subsets. Our deconvolution strategy could estimate the presence of cancer cells, stromal cells, as well as the presence of different immune cells, so our technic could give estimations of absolute numbers of immune cells. Our study confirmed that GBM are poorly infiltrated with the different types of CD8 T cells but also underlined the high presence of monocytic-like cells in mesenchymal tumours. These cells may be reminiscent of the MDSCs, which are strong immunosuppressive cells in various cancers.37 Treg and Th2, two CD4 populations with mainly protumoural properties, are the most frequent T cells in GBM. Together such data strongly support that GBM is mostly associated with protumoural and immunosuppressive cells and consequently may not be a good target for checkpoint inhibitor alone.38 Many addition works are required to determine if elimination of immunosuppressive cells improve outcome or favour infiltration by CD8 T cells and checkpoint efficacy. Surprisingly, when studied as continuous variables only a few immune cells were associated with outcome in the two cohorts, thus suggesting that immune reaction has a minor role in patients’ survival.Previous reports analysed the role of immune infiltrates in GBM with conflicting results. The prognosis roles of CD4 and CD8 infiltrates in GBM were previously reported in few small retrospective studies. Upon immunohistology, it was reported39 Recently, IDH mutation, TERT mutation and MGMT methylation allowed better stratification of patients.42 Currently, molecular signatures are not used to address prognosis. We proposed that our molecular and immune signature could be used to address patient prognosis. Limitations of our study include that this analysis was performed on Affymetrix Chips and further works are required to determine if such signatures could be performed using other technologies like NanoString or RNA sequencing, which would be more convenient in routine practice. An additional limitation of this strategy is that cell lines are not the original tumour cell and some transcriptional modification could occur in vitro and may limit interpretation of our results. Another issue in such work is that the process of deconvoluting the stroma in terms of cell type is dependent on the sample. Additional works are required to compare the results of deconvolution in multiple sample of a same tumour. Addition works will be required to determine if such signatures are only prognostic or predictive of efficacy of defined therapies. However, this observation was performed on a large cohort and externally validated in another cohort thus improving the strength of our observations.Finally, few variables could be used to better classify patients in function of their prognosis. The most powerful variables remained clinical variables including sex, age, the type of tumour resection.In conclusion, we demonstrated that GBM cell lines classification and immune classification could be combined to address GBM patient prognostic and could be used to stratify patients for further clinical trials. Immune classification may have some interest in the future. In addition to the determination of the prognosis such data was give some clues to select patients with T cells infiltrates which may gain benefit from checkpoint inhibitor. Such classification may also help us to find tumour, which need to target immunosuppression like Treg or TAM or tumour with immune desert, which could gain benefit from tumour vaccine.Supplementary table 1Supplementary table 2Supplementary table 3Supplementary methodsLegends supplementary figuresSupplementary Figures


BRAF-mutant melanoma, the MEK inhibitor (MEKi) trametinib has failed to show clinical benefit in KRAS-mutant colorectal cancer. To identify mechanisms of resistance to MEKi, we employed a pharmacogenomic analysis of MEKi-sensitive versus MEKi-resistant colorectal cancer cell lines. Strikingly, interferon- and inflammatory-related gene sets were enriched in cell lines exhibiting intrinsic and acquired resistance to MEK inhibition. The bromodomain inhibitor JQ1 suppressed interferon-stimulated gene (ISG) expression and in combination with MEK inhibitors displayed synergistic effects and induced apoptosis in MEKi-resistant colorectal cancer cell lines. ISG expression was confirmed in patient-derived organoid models, which displayed resistance to trametinib and were resensitized by JQ1 co-treatment. In in vivo models of colorectal cancer, combination treatment significantly suppressed tumor growth. Our findings provide a novel explanation for the limited response to MEK inhibitors in KRAS-mutant colorectal cancer, known for its inflammatory nature. Moreover, the high expression of ISGs was associated with significantly reduced survival of colorectal cancer patients. Excitingly, we have identified novel therapeutic opportunities to overcome intrinsic and acquired resistance to MEK inhibition in colorectal cancer.Despite showing clinical activity in  APC and TP53 and mutational activation of KRAS [Apc and Trp53 loss and KrasG12D expression in colonic intestinal epithelial cells demonstrated this by recapitulating the progression from adenoma to carcinoma, with a key role of KrasG12D being to accelerate tumorigenesis and increase the incidence of metastatic disease [KrasG12D in tumors caused them to revert to adenomas, underscoring their continued dependence on mutant Kras and providing further confirmation that Kras signaling remains an important driver of late-stage disease. Increasing evidence implicates oncogenic Ras in the modulation of the tumor microenvironment to support tumor growth [Common genetic alterations responsible for the development and progression of colorectal cancer (CRC) include inactivation of the tumor suppressors  of KRAS , 2. A rer growth , 5. Thisr growth , 7. Notar growth , 8, 9. HKRAS wild-type cancer [BRAF-mutant melanoma [RAS-mutant cancers, including CRC [Current approved targeted therapies for CRC include anti-angiogenic drugs, such as bevacizumab and regorafenib, as well as epidermal growth factor receptor inhibitors cetuximab and panitumumab for e cancer –13. The e cancer . This womelanoma . Howeverding CRC .KRAS-mutant CRC cell lines with differing sensitivity to pharmacologic MEK inhibition and identified transcriptional states associated with resistance. We demonstrate a striking enrichment of interferon- and inflammation-regulated genes in MEKi-resistant cell lines and importantly, we further associate these transcriptional states to the development of acquired resistance to MEK inhibition. Moreover, we describe in colorectal cell lines, organoids from metastatic patient samples and in xenograft and syngeneic models, a therapeutic strategy to suppress inflammatory gene expression, restore sensitivity to MEK inhibition and forestall the emergence of drug-resistant populations.Resistance to MEKis has been attributed to mutation of the drug-binding site of MEK , or throKRAS-mutant, CRC cell lines that were either sensitive or resistant to MEK inhibition. Utilizing the Cancer Cell Line Encyclopedia (CCLE), we classified the 13 cell lines based on their GI50 to the second-generation MEKi PD0325901 [50 > 8 µmol/L) and nine were classified as sensitive (GI50 < 250 nmol/L). We used comparative marker selection to identify genes that were differentially expressed between the two groups and focussed on the 140 genes that showed increased expression in the resistant cell lines by a factor of two fold or greater   Figure . We asse) Figure , Fig S2.) Figure . We also) Figure . GSEA an) Figure . Overall50 for trametinib compared with the parental cell line  and NFκB-driven super enhancers [Given that inflammatory gene expression appeared to associate with resistance to MEK inhibition, we hypothesized that its suppression may restore sensitivity to trametinib in resistant cell lines. The bromodomain inhibitor JQ1 inhibits inflammatory gene expression by the suppression of inflammatory gene super enhancers and via inhibition of NFκB p65  cultures from KRAS-mutant CRCs [MX1, IFI44L, IL1α, IL2, and TNFα , versus 52.5 d with trametinib alone, 42 d with JQ1 alone (both not significant) when compared with 44.5 d with vehicle  but this was not significantly better than trametinib alone (p = 0.4357). To confirm the efficacy of this combination in an immunocompetent model, we used the Kras-mutant, CT26 mouse syngeneic model in BALB/C mice. Whereas trametinib and JQ1 failed to slow tumor growth, the combination of both agents markedly suppressed tumor growth over 14 d  or by direct effects on immune cells, we assessed immune cell populations within the CT26 tumors by multi-color flow cytometry following 14 d of dosing  or the Deutsche Sammlung von Mikroorganismen und Zellkulturen  and grown in the recommended culture medium, supplemented with 10% fetal bovine serum, at 37 °C and an atmosphere of 5% COescribed . KRAS muquencing . InhibitCell lysis and immunoblotting techniques were as previously described , 49. TheCell proliferation assays were as previously described and quantified using CellTiter-Blue  . The druCells were treated with either DMSO or the indicated compounds. After 72 h, cells were stained with annexin V and propidium iodide using the Annexin V Apoptosis Detection Kit  and analyzed by flow cytometry .http://pga.mgh.harvard.edu/primerbank)  and reverse transcribed using the high capacity complementary DNA reverse-transcription kit . The PCR was performed using the Fast SYBR Green Master Mix (Applied Biosystems) on a ViiA 7 Real-Time PCR System (Applied Biosystems). Primer combinations were designed using the Harvard Primer Bank (k) Table .Total RNA was isolated using the miRNeasy kit . RNA samples were quality controlled and sequenced by the Tumor Profiling Unit of the Institute of Cancer Research . NEB  polyA kit was used to select the mRNA. Strand-specific libraries were generated from the mRNA using the NEB ultra directional kit. Illumina paired-end libraries were sequenced on a HiSeq2500  using v4 chemistry acquiring 2 × 100 bp reads. Bcl2fastq software  was used for converting the raw base calls to fastqs and to further demultiplex the sequencing data. The paired-end fastq files were used for further analysis. Tophat2 spliced alignment software was used to align reads to the reference genome (GRCH37) in combination with Bowtie2. Once the reads were aligned, HTSeq-count was used to count the number of reads mapping unambiguously to genomic features in each sample. Differential expression analysis of the count data was done in R using the DESeq2 Bioconductor package. The lists of up- and downregulated differentially expressed genes were then tested for enrichment of gene sets uniquely defining the previously defined CRC subtypes  using thBRD4 (L-004937-00-0005), NFkB p65 (L-003533-00-0005), IFIT1 (L-019616-00-0005), and MYC (L-003282-02-005)  and a non-targeting siNT-control (D-001810-01-05) were pre-incubated with Lipofectamine RNAiMAX  and Opti-MEM culture medium  according to the manufacturer’s instructions. Cells were reverse transfected with the siRNA-lipid complexes and incubated at 37 °C for the indicated time points until further analysis.siRNAs targeting 6 cells into the right flank of female NCr mice and randomly allocated into treatment groups. Treatment using published, efficacious schedules was initiated when tumors reached a mean diameter of ~6 mm (indicated as day 0). Control mice (n = 10) received vehicle (1% Hydroxypropylbetacyclodextrin (2-hydroxypropyl)-β-cyclodextrin) po, 10% DMSO in 10% w/v Hydroxypropylbetacyclodextrin ip), and treated mice (n = 10) were given 1 mg/kg trametinib orally or 50 mg/kg JQ1 administrated by intraperitoneal injection or the combination of both drugs daily for of 28 d [3, and body weights were determined three times weekly. A dosing holiday was given to all groups on day 21 to aid tolerability. CT26 tumors were established by injection of 5 × 105 cells into the right flank of female BALB/C mice and treated as above. All animal studies were approved by the local research ethics committee and carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and National Guidelines [T84 tumors were established by subcutaneous injection of 5 × 10 of 28 d , 52. Tumidelines . ApproprTumors were dissociated into a single-cell suspension using a gentleMACS Octo Dissociator with Heaters  and the Mouse Tumor Dissociation Kit (Miltenyi Biotec). Samples were run on the 37C_m_TDK_1 program, applied to a 70 μm MACS SmartStrainer and washed in phosphate-buffered saline (PBS). Erythrocytes were removed from samples by suspension in red blood cell lysis buffer for 5 min at room temperature. Samples were resuspended in PBS for flow cytometry staining.Cells were stained with a fixable viability dye (Thermo Fisher Scientific) and blocked with an anti-mouse CD16/CD32 antibody (Thermo Fisher Scientific). A panel of fluorescence-conjugated antibodies was added to cell suspensions at specified dilutions Table  and incuSupplementary materialSupplementary tables


Tellurium is a rare element that has been regarded as a toxic, nonessential element, and its biological role is not clearly established. In addition, the biological effects of elemental tellurium and some of its organic and inorganic derivatives have been studied, leading to a set of interesting and promising applications. Diphenyl ditelluride (DPDT), an organic tellurium derivate, showed antioxidant, antigenotoxic, antimutagenic, and anticancer properties. The antioxidant and prooxidant properties of DPDT are complex and depend on experimental conditions, which may explain the contradictory reports of these properties. In addition, DPDT may exert its effects through different pathways, including distinct ones to those responsible for chemotherapy resistance phenotypes: transcription factors, membrane receptors, adhesion, structural molecules, cell cycle regulatory components, and apoptosis pathways. This review aims to present recent advances in our understanding of the biological effects, therapeutic potential, and safety of DPDT treatment. Moreover, original results demonstrating the cytotoxic effects of DPDT in different mammalian cell lines and systems biology analysis are included, and emerging approaches for possible future applications are inferred. Tellurium (Te) is a stable and solid element that pertains to chalcogens (group 16 in the periodic table), which is the same group that includes sulfur, selenium, and polonium. Te is classified as a metalloid because of its features between metals and nonmetals . It was The industrial applications of inorganic Te compounds include production of nanoparticulate semiconductors and metal-oxidizing solutions , 7. FurtThe risk of human environmental exposure to Te is unpredictable due to its elevated usage . The useAlthough there was limited use of synthetic organotellurium (OT) compounds in the past, they have turned a promising alternative for various applications, as evidenced by the increase in reports on OT compounds in the literature , 19. SynIn the present review, we emphasize the biological activities of an OT compound, diphenyl ditelluride (DPDT) , aiming The antioxidant effects of certain molecules are based on their ability to retard or inhibit oxidative damage. Their antioxidant role includes blocking oxidative reactions induced by highly reactive oxidant molecules—the so-called free radicals or reactive oxygen species (ROS)—that damage other molecules. The antioxidant properties of substances such as OT compounds can protect the biomolecules and cell components against oxidative damage –26. OT cin vitro studies and rodent models could be attributed to their ROS scavenging and glutathione peroxidase mimetic properties . Th. ThμM DP5 μg/mL) .μg/mL DPDT for all cell lines. A DPDT concentration of 0.28 μg/mL induced a greater reduction in cell viability, about 40–50%, for all cell lines  was evaluated by clonogenic assay. The cells were treated for 72 h at 0.028, 0.28, and 2.8 ll lines . These rThe administration of traditional chemotherapeutic agents inhibits the cell division inducing not only cell cycle alterations and apoptosis but other forms of nonapoptotic cell death such as necrosis, autophagy, and mitotic catastrophe. Since the most effective cancer treatment method used after surgery is chemotherapy, the search for new drugs with antiproliferative properties is currently ongoing , 55.μM DPDT. This was the first study showing the effects of DPDT on the cell cycle (μM DPDT resulted in the accumulation of S-phaseV79 cells . In. InμM DP79 cells . Moreovell cycle . We alsoll lines . FurtherC6 cells .μM) (μM) in V79 cells . In. InμM)  showed pronounced tolerance in the top1Δ strain to DPDT exposure. The same study also reported DPDT-induced inhibition of human TopoI activity in vitro using DNA relaxation assays [The results reported in DPDT studies lead us to infer the possible mechanisms of action of this drug and suggest its application as an antiproliferative agent in cancer therapy. As previously mentioned, results from our group demonstrated that DPDT can induce frameshift mutations in bacterial DNA and induce the formation of micronuclei in V79 cells , 22. On 79 cells . In thisn assays . These rRegarding the possibility of TopoI inhibition, the search for a noncamptothecin (CPT) TopoI inhibitor has been the target of several studies because of the limitation of CPT and its derivatives –62. The The intertwining of DNA strands and helices, produced during the essential cellular processes of replication, transcription, recombination, and chromosome segregation, must be resolved in order to maintain genome stability and cell viability. DNA topoisomerases supply an important solution for resolving such topological DNA problems . Howeverp value ≤ 0.01. After this, we obtained initial C6 protein-protein interaction (PPI) networks based on physical interactions from the GeneMANIA prediction server [As described in previous sections, DPDT promotes different cellular phenomena. However, no molecular target associated with DPDT has yet been identified. We used a systems biology approach to predict putative cell signaling pathways or specific protein targets for DPDT. To this end, we prospected networks based on C6 cell line gene expression data. Contrast analysis was applied, and differentially expressed genes before simulation of DPDT treatment were selected using the rank product method, a technique based on calculating rank products (RP) from replicate experiments. We used three sample replicates of the C6 rat glioma cell line (untreated) from the GEO database (GSE1139 accession number) under culture conditions similar to those of our experimental model. For each sample, the average of the signal between the same probes was calculated and applied to the normalized microarray data using the limma package in the R/Bioconductor software , 72. Then server  which aln server . It is an server –77.In addition, to define Akt interconnectivity and relevance in the C6 CP-PPI network, we analyzed some topological parameters. Firstly, we applied CentiScaPe software to explore node degree and betweenness centralities . Based o2+ influx activated protein kinase A and protein kinase C in astrocytes, causing the hyperphosphorylation of glial fibrillary acidic protein and vimentin. These disrupt the organization of actin stress fibres formed by endogenous RhoA activation and led to altered cell morphology. In neurons, the overexpression Ca2+ levels activated Erk and p38MAPK, beyond the protein kinase A and protein kinase C, provoking hyperphosphorylation of neurofilament subunits  was observed at a similar concentration range to that of the antitumor agent DOX (IC50: 0.02 μg/mL). This concentration is more than an order of magnitude lower compared to the toxic DPDT concentration in normal CMSPH cells (0.4 μg/mL). Furthermore, the hemolytic potential in erythrocytes was observed at high concentration, and the cytotoxicity of DPDT cannot be attributed to unspecific damage to cell membranes. Albeit the conventional prejudice concerning organotellurium compounds, DPDT use should be considered with caution because of its high reactivity and toxicity at relatively low concentrations. The exact nature of DPDT cytotoxic effects remains unclear, although some mechanisms have been proposed that differ depending on the system studied. New trials regarding toxicity, checkpoint activation, and mechanisms of cell death induction by DPDT should be explored in a greater number of cell lines. Moreover, its topoisomerase inhibition potential should be further investigated, keeping in mind that this compound probably presents several distinct mechanisms of action. DPDT have particular chemistry with the thiol which is related to many of the biological effects observed so far. The DPDT depletes GSH because of oxidation and/or as a possible substrate for GSH conjugation and could modulate cellular antioxidant defenses inducing GSH synthesis. The dual action of DPDT (protective and toxic) opens possibilities for distinct applications in cancer treatment (2+ levels activated Erk and p38MAPK, beyond the PKA and PKC, provoking hyperphosphorylation of neurofilament subunits, and as a consequence must have caused cellular redox imbalance, increasing ROS and inducing cell death. ROS-generating drugs induce cancer-specific cytotoxicity by elevated endogenous ROS production in preclinical studies [In the past decade, several organometallic compounds have entered clinical trial owing to their unique redox-modulating features and great potential in cancer therapy , 81. Thereatment . In neur studies , 83. Thu


The results showed that after 24 h of lectin exposure, the cells lines showed a dose-dependent cytotoxic effect, the effect being higher on MCF-7, while C33-A showed the highest resistance. Cell proliferation studies showed that the toxic effect induced by lectins is higher even when lectins are removed, and in fact, the inhibition of proliferation continues after 48 h. Due to the use of two techniques to analyze the cytotoxic and antiproliferative effect, differences were observed in the results, which can be explained by the fact that one technique is based on metabolic reactions, while the other is based on the 3[H]-thymidine incorporated in DNA by cells under division. These results allow concluding that lectins exert a cytotoxic effect after 24 h of exposure, exhibiting a dose-dependent effect. In some cases, the cytotoxic effect is higher even when the lectins are eliminated, however, in other cases, the cells showed a proliferative effect.For many years, several studies have been employing lectin from vegetables in order to prove its toxic effect on various cell lines. In this work, we analyzed the cytotoxic, antiproliferative, and post-incubatory effect of pure tepary bean lectins on four lines of malignant cells: C33-A; MCF-7; SKNSH, and SW480. The tests were carried out employing MTT and  Worldwide cancer-related mortality has increased considerably, thus the need to search for novel drugs and alternative treatments to solve this health problem. One of these alternatives has suggested the use of diverse alternative treatments such as herbal medicines, something that has been known for many decades by humans ,2,3.in vitro and in vivo, inhibit growth when incorporated into the diet, and are toxic when injected into animals; therefore, it has been suggested that lectins be utilized as an alternative treatment for some diseases, such as cancer -thymidine assays.Tepary bean -2,5 diphenyltetrazolium bromide (MTT) assay. The MTT assay is a colorimetric assay for assessing cell viability, however, tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferative to resting status) of potential medicinal agents and toxic materials.The cytotoxicity of the TBL to SKNSH, SW480, MCF-7, and C33-A cells after 24 h of exposure is depicted in Studies carried out employing tritium-labeled thymidine shown in  indicate3[H]-thymidine assays.To establish whether the effect of lectin toxicity continues when this substance has been eliminated from the cell culture, we carried out another assay in which, after a 24-h lectin exposure, these proteins were removed and cytotoxicity and proliferation was again determined after 24 and 48 h of incubation with only culture medium, employing MTT and The results obtained by MTT assay are depicted in These results indicated that once the lectin was removed from the cells, the cytotoxic effect of these proteins continued, causing the cells to inhibit their proliferation after 24 h of incubation, which at this point indicated that the lectins were attached to the cell membranes. This prevents the cells from being able to metabolize the MTT, which is reflected in the decreased production of the formazan compound. However, at this point, we are unable to know with certainty whether the cells are able to maintain this effect, thereby continuing cell incubation without lectin for an additional 24 h, up to 48 h.When the cell proliferations were analyzed after 48 h of incubation without lectin, the results illustrated in As can be observed, some of the cell lines continued to decrease in viability; SKNSH cells, however, were able to recover and increase their cell viability. These results may indicate that although the lectin caused damage, these cells are able to repair this damage and to continue to perform certain metabolic functions, or that the level of damage caused by the lectins was not permanent and that cell proliferation could be recovered at some time after the lectins were removed from the culture medium. However, on the other hand, in the remaining cell lines, damage was more severe, perhaps because the lectins were more strongly bound to the cells and were unable to recover.The post-incubatory assay carried out employing is shown in The results when proliferation was measured after 48 h without lectins are illustrated in These results indicated that at low concentrations, the cytotoxic effect of lectins was maintained, even if these were not still present in the culture medium. However, in lectins that is bound strongly the damage continue although the lectin solution was retired, but at higher concentrations of 100 µg/mL, recovery cells exhibited activity, thus an increase in the ability to divide by increasing the amount of thymidine incorporated into the DNA.Many studies have reported the purification of the lectins of several vegetable sources, including ion exchange and affinity ,52,53,54During lectin purification by affinity chromatography, it was determined that the fraction not retained by the matrix continued to demonstrate considerably high agglutination activity. This is probably due to that the fetuin matrix does not possess the ability to retain some of the lectin isoforms contained in the beans, retaining in this manner only a fraction of these. There are reports that indicate that lectins of different bean varieties consist of several isoforms and that in order to purify these independently, it is necessary to employ certain chromatography techniques, either independently or in combination, such as ion exchange or affinity, because each isoform possesses a different purification pattern ,56,57.When we analyzed the pure lectin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (data not shown), this exhibited only one band with a molecular weight of approximately 31 KDa, which led us to think that only one protein was purified. However, previously published studies in which this same affinity matrix was employed showed that such a matrix not only retains a single isoform, but also receives a mixture of all three of these, which differ little in their isoelectric points and molecular weights . AnotherThe MTT technique is known as an assay that is based on the ability of living cells to transform 3--2,5-diphenyltetrazolium bromide (MTT) by means of the action of the mitochondria into a colorful formazan product, which is a direct indicator of the amount of viable cells, and also can be used to measure cytotoxicity or cytostatic activity (shift from proliferative to resting status); however, there was a degree of error, due to that there may be cells that do not exhibit immediate metabolic activity or even that their activity is decreased due to the action of the lectin bonding to the cells. Although this methodology is well accepted for carrying out feasibility and cell proliferation studies, there are other methodologies that can yield better results concerning the cells’ biological activity, such as the technique that includes DNA synthesis, as well as the use of tritiated thymidine.The results observed in the study with the MTT assay indicate that there is great variability in the cytotoxic effect of lectins on the four cell lines studied. It is noteworthy that these cell lines derive from different cancer origins, but it has been documented that there are significant changes in the pattern of the glycosylation of cell membranes in the carcinogenic process. The binding of lectins to cell membranes depends on the type of carbohydrate to which the lectin is related, and also to that found in the cell membrane and, on the other hand, to the strength of the binding of these lectins. As we can observe, although in all cell lines the lectins were most likely bound by the union pattern, this was considerably variable and the quantity of lectins bound was also different, all of this due to the differences of each cell line, as well as to that the damage caused by the lectin in each cell line is varied, despite that statistically, not a great difference has been noted among the lines. As mentioned previously, the cell line that exhibited the greatest effect was SW480; this could have been due to that lectin bound in a greater quantity and exerted a greater toxic effect on these cells, in contrast with the remaining cell lines studied.With the use of the tritiated thymidine technique, we are very well aware that this is a much more accurate technique than the MTT one for directly measuring the amount of thymidine incorporated by living cells and their capability of dividing, which renders it a better technique for measuring lectin-associated cellular damage. Thus, this technique more clearly shows the level of damage caused by the lectins in each of the cell lines.in vivo, causing damage to several cells in organs -Thymidine Assay4.3.2. 4 cells/well and incubated at different TBL concentrations  in DMEM for 24 h. After a 24 h exposure to the lectin, the solution was removed, and DMEM supplemented with FBS was added. Incubation was continued for 24 and 48 h and cell proliferation was measured at each period by detection of the incorporation of tritium-labeled thymidine (3 µCi/well) during a 30 min incubation period. To stop the uptake of radioactivity, the medium was removed and the cells were washed twice with phosphate buffer pH 7.6 containing 150 mM NaCl; then, we added 500 µL of SDS 0.1% containing 10 mM ethylenediaminetetraacetic acid  to each well. After 30 min at room temperature, the lysate was added with 500 µL of 10% cold trichloroacetic acid (TCA) and the precipitate was collected on a nitrocellulose paper, washed three times with TCA 5%, dried, and counted in a liquid scintillation counter . All determinations were performed in triplicate.The cells were cultured in 24-well flat-bottomed plates at a concentration of 5 × 10p < 0.05), followed by the Tukey post hoc test for multiple comparisons. IC50 values were calculated from linear regression analysis.Experimental results are expressed as mean ± SEM. All measurements were replicated three times. The data were analyzed by an analysis of variance (Purification of lectins from tepary beans using the fetuin affinity column was a good choice for purification, however, purification could be improved by employing another affinity matrix with higher affinity for tepary lectins, or some combination with other chromatographic techniques, to improve lectin purification and separate the lectin isoforms. We observed a marked effect of the lectins on the inhibition of cell proliferation in the SW480 cell line, with the effect not being so marked in the remaining three cell lines studied.The post-incubation results obtained 24 and 48 h after the lectin was eliminated from the cell culture showed that by the MTT technique, the four cell lines studied possessed some recovery capacity, displayed by increasing cell proliferation. On the other hand, post-incubation proliferation results obtained by tritium-labeled thymidine after 24 and 48 h prior to the lectin solution being eliminated from the cell culture showed that SW480 cells were not able to recover their proliferation activity: the C33-A and MCF-7 cell lines did not present recovery at low lectin concentrations, while at high concentrations, both cell lines presented recovery. In the case of SKNSH cells, these demonstrated proliferation activity after elimination of the lectin. Recovery in cell proliferation 24 and 48 h after the lectin was eliminated, as observed in SKNSH, C33-A, and MCF-7 cell lines, was highest at the 100-µg/mL concentration.


Aeschynomene fascicularis (Fabaceae) has been used in Mayan traditional medicine in the Yucatan peninsula. However, the compounds present in the plant responsible for its curative properties have not yet been investigated. Aeschynomene fascicularis root bark was extracted with 100% methanol to obtain a crude extract. The methanol extract was partitioned successively with solvents with increasing polarity to obtain the corresponding hexane (Hx), dichloromethane (DCM) and ethyl acetate fractions (EtOAc), as well as a residual water-alcoholic fraction. These fractions were tested for their cytotoxic activities using an MTT assay against Hep-2 cancer cell lines. The Hx fraction led to the isolation of spinochalcone C (1), spinochalcone A (2), isocordoin (3) and secundiflorol G (4). Their structures were identified based on spectroscopic evidence and chemical properties. All compounds were subjected to cytotoxicity and antiproliferative assays against a panel of seven cell lines, including one normal-type cell line. Spinochalcone A (2) exhibited cytotoxic activity against DU-145 cell line and antiproliferative activity against the KB cell line. Secundiflorol G (4) showed strong cytotoxic activity towards KB and Hep-2 cell lines. In addition, isocordoin (3) showed moderate activity on KB, Hep-2 and DU-145 cell lines. The active Compounds 2, 3 and 4 are potential therapeutic entities against cancer.The plant  The flora of the Yucatan peninsula is rich in vascular plants, including 2600–3000 species . Some ofAeschynomene fascicularis root bark has a pronounced cytotoxic activity on cancer cell lines.In the Yucatan, medicinal plants have been used in urban and rural communities as a common practice for the control of many types of diseases, including cancer. Moreover, they are used in the treatment of conditions consistent with the symptoms of cancer: abscesses, calluses, corns, hard lumps, polyps, tumors or warts. In a preliminary screening for cytotoxic constituents from plants used in Mayan traditional medicine to treat cancer-like symptoms, 21 species were collected, and the crude methanol extracts of various plant parts were subjected to a cytotoxicity assay against a panel of cancer cell lines . The resAeschynomene fascicularis Cham. and Schltdl. (Fabaceae) is a shrub extensively distributed from Mexico to Colombia. This plant is commonly known as kabal pich by the ancient inhabitants of the Yucatan Peninsula, and it has been used in Mayan traditional medicine to treat superficial tumors [Aeschynomene genus have been done [A. fascicularis [A. fascicularis.l tumors . Few cheeen done . Recentlicularis . TherefoA. fascicularis.Continuing our effort to search for novel anti-cancer agents from Mayan medicinal plants of the Yucatan peninsula, we performed a guided fractionation of the root bark extract to isolate and identify the cytotoxic compound(s) from this plant. Thus, the aim of this study was to isolate and identify the active cytotoxic compounds from A. fascicularis root bark was subjected to chromatographic purification, resulting in the isolation of four compounds. The chemical structures of all compounds were determined by the analysis of their NMR spectra  and mass spectra and by comparison of these data with those published in the literature.The active hexane fraction obtained from the methanol extract of 1: yellow oil. UV λmax (CHCl3) nm: 239, 308; FTIR (film) νmax cm−1: 3385, 1649, 1600, 1450, 1332, 1122; EI-MS 70 eV, m/z (relative intensity %): 374 , 359 (100), 303 (25), 255 (20), 131 (15), 103 (17). For 1H-NMR  and 13C-NMR  data, see 1 showed a molecular mass of 374 a.m.u. Its FTIR spectrum showed bands at 3385 and 1649 cm−1, which in conjunction with the 1H-NMR signal of a chelated proton at δ 13.74 , signals due to two trans-olefinic protons at δ 7.57  and δ 7.86 , and an α,β-unsaturated carbonyl signal at δ 191.9 in the 13C-NMR spectrum, confirmed the structure of a chalcone. Additionally, the 1H-NMR spectrum showed signals in the aromatic region belonging to a singlet at δ 7.40 and two sets of multiplets at δ 7.43 (3H) and δ 7.65 (2H) characteristic of protons in penta-substituted and mono-substituted rings, respectively. The presence of two cis-olefinic protons at δ 5.59  and δ 6.32  along with two signals for two methyls (δ 25.9 and 18.0) and a signal at δ 77.8 due to a quaternary carbon in the 13C-NMR spectrum are consistent with the occurrence of a 2,2-dimethyl-pyran ring. Furthermore, signals at δ 1.46 (6H), δ 3.36  and a multiplet at δ 5.26 (1H) indicate the presence of an isoprenyl group. The position of all protons and carbons was established by combining the two-dimensional HSQC and HMBC experiments. The assigned structure (Tephrosia spinosa [Compound tructure  correspo2: yellow amorphous solid. UV λmax (CHCl3) nm: 238, 322; FTIR (film) νmax cm−1: 3390, 1634, 1608, 1567, 1362, 1147; 1H-  and 13C-  NMR: see m/z (relative intensity %): 376 , 333 (40), 305 (45), 265 (30), 201 (40), 161 (60), 131 (40), 103 (35). Compound 2 has a molecular mass of 376 a.m.u. A set of signals in the 1H, 13C-NMR and FTIR spectra indicated the presence of a chalcone skeleton similar to Compound 1. However, 2 presents two isoprenyl groups confirmed by the presence of methyl signals at δ 1.76, 1.79, 1.80 and 1.84 (3H each), two signals at δ 3.33 and 3.46  and a multiplet at δ 5.30 (2H). The location of all protons and carbons was established by combining the two-dimensional HSQC and HMBC experiments. The assigned structure (Tephrosia spinosa [1 and 2 are reported here for the first time.Compound tructure  correspo3: yellow amorphous solid. FTIR (film) νmax cm−1: 3231, 1629, 1613, 1480, 1444, 1362, 1234; EI-MS 70 eV, m/z (relative intensity %): 308 , 265 (90), 231 (12), 149 (100), 131 (37), 103 (40), 77 (20). 1H-NMR : δ 1.78 , 1.85 , 3.49 , 5.31 , 6.44 , 7.43 , 7.60 , 7.74 , 7.89 , 13.76 . 13C-NMR : δ 18.2 (3′′-Me), 22.0 (C-1′′), 26.0 (3′′-Me), 108.1 (C-5′), 114.3 (C-1′), 114.4 (C-3′), 120.8 (C-α), 121.3 (C-2′′), 128.7 (C-2/C-6), 129.2 (C-3/C-5), 129.6 (C-6′), 130.8 (C-4), 135.1 (C-1) 136.1 (C-3′′), 144.5 (C-β), 162.0 (C-4′), 164.2 (C-2′), 192.3 (C=O). Compound 3 gave identical spectral features with those described for isocordoin, which has previously been isolated from Cordoa piaca [Lonchocarpus xuul [Compound 4: brown-red amorphous solid. c 0.02, CHCl3); UV λmax (CHCl3) nm: 238, 283; FTIR (film) νmax cm−1: 3385, 1618, 1598, 1506, 1454, 1275, 1157, 1116. In the 1H-NMR spectrum, a set of mutually-coupled five protons , 3.02 , 3.52 , 4.09  and 4.36 ) suggested the presence of an isoflavan skeleton. The 1H-NMR spectrum further exhibited the presence of a methoxyl at δ 3.74 (s), and signals of a typical α,α-dimethyl-allyl group were observed , 1.51 , 4.97 , 5.02 , and 6.13 . An isolated aromatic proton at δ 6.56  and three protons in an ABC system , 6.38  and 6.92 ) indicate that an aromatic ring is penta-substituted, and another ring is tri-substituted. Characterization of Compound 4 was made by comparing the 1H-NMR spectrum to that reported for secundiflorol G, a flavan previously described as isolated from Sophora secundiflora [Aeschynomene and, particularly, on the contribution to the phytochemistry of the species A. fascicularis.Compound A. fascicularis. Our guided fractionation study suggests that flavan 4 and chalcones 2 and 3 are the main compounds responsible for the observed biological activity in the original extract and the active hexane fraction. The results of the bioassays towards six human cancer cell lines and their selectivity indices (SI) compared with a normal cell line are presented in This study was conducted towards the isolation of active compounds from the root bark of 1 did not show any activity on tested cell lines to the maximum concentration (100 µM).Compound 2 showed good cytotoxic activity against the DU-145 and PC-3 cell line; moreover, this compound showed high selectivity compared with a normal cell line. Interestingly, only this compound showed good antiproliferative activity toward the KB cell line  towards all tested cell lines. Cytotoxic activity of isocordoin (3) on ovarian, prostate and leukemia human cancer cell lines has been documented [Leishmania mexicana and Trypanosoma cruzi [Compound cumented . Further4) showed potent activity and moderate selectivity towards oral-laryngeal cancer cell lines Hep-2 and KB. Previously, the cytotoxic activity of various isoflavans had been reported against mouth cancer cell lines [4) towards KB and Hep-2 lines. Therefore, we propose the potential use of secundiflorol G in oral-laryngeal cancer chemotherapy.Secundiflorol G  is the most abundant compound present, as much as 30% of the hexane fraction of A. fascicularis root bark (measured by GC-MS), and this could be exploited to develop 2 as a potential marker molecule (together with 3 and 4) to standardize the hexane fraction of A. fascicularis as a phytomedicine. In addition to this, it is worth highlighting the antiproliferative effect that 2 presented against the KB cell line, providing an added value to the development of A. fascicularis as a phytomedicine for the treatment of oral-laryngeal cancer. At present, cancer therapies are using multiple cytotoxic drugs acting synergistically, in order to enhance the curative potency of all substances together. This emphasizes the effectiveness of a phytomedicine prepared from the hexane fraction of A. fascicularis.Finally, spinochalcone A . The FTIR spectra were measured with a Nicolet Protégé spectrophotometer . NMR spectra  were acquired on a Bruker Avance  400 Ultra Shield spectrometer and TMS was used as an internal standard. Low-resolution mass spectra were taken on an Agilent Technologies  6890N gas chromatograph coupled to a 5975B mass spectrometer (GC-MS). Column chromatography was carried out on silica gel 60 (70–230 mesh) from Merck  and Sephadex LH-20 from Sigma . TLC was performed on precoated silica gel 60 F254 (0.25 mm) (Merck). Spots on TLC were visualized under UV light and by spraying phosphomolybdic acid reagent, followed by heating. The optical density of the developed plates with the 3--2,5-diphenyl tetrazolium bromide  and sulforhodamine B  was read in a ThermoSpectronic spectrophotometer from Bio-Rad .UV spectra were taken in CHA. fascicularis was collected in May 2009 from Yaxcabá, Yucatan  (Mexico). The plant material was identified and authenticated by a taxonomist from the Natural Resources Unit of the Centro de Investigación Científica de Yucatán (CICY). Specimens under the voucher number P. Simá 2997 were deposited at CICY’s U Najil Tikin Xiw herbarium.Root bark of 2O (1:3) and extracted using solvents of increasing polarity (3 × 1000 mL): hexane, CH2Cl2 and EtOAc. The solvents were removed using a rotary evaporator (40 °C) to obtain the hexane, dichloromethane, ethyl acetate and aqueous fractions. Only the hexane fraction showed cytotoxic activity against the Hep-2 cancer cell line (CC50 = 18 µg/mL).Dried powdered root bark (594 g) was extracted by maceration at room temperature with MeOH (3 × 500 mL) for 24 h. The supernatants were filtered and evaporated under vacuum by means of a rotary evaporator to obtain a dried extract. The MeOH extract (84 g) was suspended in 500 mL MeOH/H1, 60 mg) and spinochalcone A , respectively. Fraction A4 was explored using a Sephadex LH-20 CC to obtained four fractions (B1–B4). Fraction B2 was eluted with MeOH (100%) and was further subjected to CC silica gel using the isocratic system Hx/An (8:2) to obtain Compound 3 (5 mg). Finally, from Fraction A6, Compound 4 (20 mg) was isolated by means of a CC silica gel using the solvent system CH2Cl2/MeOH (9:1).The active hexane fraction (6.4 g) was submitted to a vacuum liquid chromatography (VLC) on silica gel using mixtures of hexane (Hx) and acetone (An) of ascending polarity to yield 11 fractions, which were pooled into six final fractions (A1–A6), according to their similarity on TLC. Fractions A2 and A3 were each purified by silica gel column chromatography (CC) under isocratic elution with Hx/An (9:1) to yield spinochalcone C (v/v), 100 U/mL penicillin G, and 100 mg/mL streptomycin at 37 °C in an atmosphere of 5% CO2 (95% humidity). At 70%–80% confluence, cells were detached from the cultured flask by treatment with 0.05% trypsin-EDTA (Gibco), and a suspension of 5 × 104 cells/mL of viable cells was seeded in a 96-well microtiter plate  and incubated.Three types of cancer cell lines obtained from the American Type Culture Collection (ATCC) were cultured: two oral-laryngeal cancer cell lines, nasopharynx carcinoma  and laryngeal carcinoma ; two cervix-uterine cancer cell lines, cervix adenocarcinoma  and cervix squamous carcinoma ; and two prostate cancer cell lines, prostate carcinoma  and prostate adenocarcinoma ; along with one normal cell line . The cells were cultured in sterile Costar T75 flasks containing DMEM , supplemented with fetal bovine serum . The amount of MTT-formazan is directly proportional to the number of living cells and was determined by measuring the optical density (OD) at 590 nm using a bioassay reader (Bio-Rad). All determinations were performed in triplicate.Cytotoxicity was assessed by the MTT assay as previously described by Rahman . When ceThe growth inhibition of cancer cells was evaluated by the sulforhodamine B method . When cecontrol − ODsample/ODcontrol) × 100. Results are expressed as the concentration of compound that killed 50% of the cells (CC50) and that reduced cell growth by 50% (IC50), respectively, and they were calculated using the software GraphPad Prism 4. In addition, the level of cytotoxicity on normal cells was evaluated by determining the selectivity index (SI) of each compound, which is calculated as the ratio of cytotoxicity or reduced cell growth on normal cells to cancer cells  [The cytotoxic and antiproliferative activities of tested compounds were calculated as the percentage of cells killed or the percentage of inhibition of cell proliferation, respectively, using the equation: growth inhibition (%) = (ODr cells) . It is gr cells) .2 and 3 and flavan 4 are the main compounds responsible for the observed biological activity in the original extract of A. fascicularis and are potential candidates for drug development based on their effective cytotoxic and antiproliferative activities.The present study suggests that chalcones