WT) and mutant type  amyotrophic lateral sclerosis (ALS) models. The uptake of [3H]L-carnitine was dependent on time, temperature, concentration, sodium, pH, and energy in both cell lines. The Michaelis–Menten constant (Km) value as well as maximum transport velocity (Vmax) indicated that the MT cell lines showed the higher affinity and lower capacity transport system, compared to that of the WT cell lines. Additionally, LC uptake was inhibited by organic cationic compounds but unaffected by organic anions. OCTN1/slc22a4 and OCTN2/slc22a5 siRNA transfection study revealed both transporters are involved in LC transport in NSC-34 cell lines. Additionally, slc22a4 and slc22a5 was significantly decreased in mouse MT models compared with that in ALS WT littermate models in the immune-reactivity study. [3H]L-Carnitine uptake and mRNA expression pattern showed the pretreatment of LC and acetyl L-carnitine (ALC) attenuated glutamate induced neurotoxicity in NSC-34 cell lines. These findings indicate that LC and ALC supplementation can prevent the neurotoxicity and neuro-inflammation induced by glutamate in motor neurons.L-Carnitine (LC) is essential for transporting fatty acids to the mitochondria for β-oxidation. This study was performed to examine the alteration of the LC transport system in wild type (WT, NSC-34/hSOD1 Amyotrophic lateral sclerosis (ALS), which is a devastating neurodegenerative disease, causes motor neuronal loss in the brain stem and spinal cord, resulting in paralysis, muscle wasting and respiratory failure, which ultimately leads to death. The demand for extending the quality of life in ALS patients is increasing, mainly due to the lack of an effective cure for this disease ,2. FamilCarnitine plays a major role in the transportation of long chain fatty acids to the mitochondria for β-oxidation, a process which generates ATP. Under physiological conditions, oxidization of fatty acids primarily involves mitochondrial β-oxidation, which is an important energy production mechanism found in mammals ,9. The mOrganic cationic/carnitine transporters (OCTN) are unique zwitter-ionic transporters expressed in mice. These are categorized under the solute carrier family (SLC) and its members 4, 5 and 21 are represented by OCTN1/Slc22a4, OCTN2/Slc22a5, and OCTN3/Slc22a21 ,13. AppaG93A mutation were used to examine the neuroprotective effects of LC and ALC. In this study, we hypothesized that carnitine and ALC pretreatment may provide neuroprotective effects against glutamate-induced excitotoxicity in ALS model cell lines. Similarly, we investigated the effects of altering carnitine transportation, as well as carnitine pretreatment, on [3H]L-carnitine uptake in motor neurons, such as NSC-34 cell lines.Previous studies have demonstrated that concentrations of various compounds, such as amino acids and monocarboxylates, are altered in the mutant ALS model. Therefore, investigating the transportation and supplementation of LC is important for acquiring a better understanding of mammalian mitochondrial energy metabolism and the prevention of neurodegenerative diseases, such as ALS ,16. Howe3H] (77.0 mCi/mmol) was purchased from Perkin-Elmer . All chemicals and reagents were high-grade commercial products purchased from Sigma-Aldrich .L-Carnitine hydrochloride L-Carnitine uptake in NSC-34 cells was studied based on a previously described method [3H]L-carnitine (32.5 nM) labeled compound was then added in the presence or absence of inhibitors for a designated time period at 37 °C -L-carnitine at 15 min and the concentration of unlabeled carnitine, respectively.For kinetic studies , the Mici) was calculated using Equation (3).For the competitive inhibition study ,20, low-® 2000 (Invitrogen). After the designated time interval, mRNA expression level and [3H]L-carnitine uptake in siRNA-transfected NSC-34 cell lines were evaluated  in a blocking solution at 4 °C for 24 h. After washing three times with PBS, the tissue slides were processed with Vector ABC Kit . The Slc22a4 and Slc22a5 signals were developed with DAB chromogen . Images were then analyzed via using an Olympus microscope system . The semi-quantification of immunoreactivity was analyzed by NIH ImageJ. Especially, the intensity of Slc22a4 and Slc22a5 signals was counted from 5~10 motor neurons per section.3H]L-carnitine uptake, and slc22a4 mRNA expression level were examined  assay was performed, following which [examined .Statistical analyses were conducted via the unpaired 2-tailed Student’s t-test or one-way ANOVA followed by Dunnett’s post-hoc test. Sigma Plot  was used to create figures.3H]L-carnitine transport system on WT and MT NSC-34 cell lines. The uptake of [3H]L-carnitine was time-and temperature-dependent until 60 min in both NSC-34 WT and MT cell lines (3H]L-carnitine transporter in NSC-34 cell lines (m1) and the maximum uptake rate (Vmax1) were 1.94 ± 0.34 µM and 0.295 ± 0.059 pmol/mg protein/min, respectively, for the WT cell line  values for WT and MT cell lines, were 0.149 (µL/(min/mg protein)) and 0.168 (µL/(min/mg protein)), respectively , and rotenone was used to deplete ATP , γ-butyrobetaine (GBB), acetyl L-carnitine (ALC) and tetraethyl ammonium (TEA) (To examine the transporter(s) responsible for transporting carnitine into NSC-34 cell lines (ALS model cell lines), cellular uptake of [um (TEA) . Similarum (TEA) .3H]L-carnitine uptake intersected at the ordinate axes, showing competitive inhibition in the presence or absence of metformin, DPH, quinidine and pyrilamine in both WT , mOCTN2 (slc22a5) and control siRNA (negative control) then using the transfected cells, the mRNA expression levels and L-carnitine uptake was evaluated . mOCTN1 n = 7) compared to normal subjects (n = 7) (n = 7) compared to normal subjects (n = 7), indicating the altered expression of carnitine transporters in human ALS patients ] mice. Interestingly, the immunoreactivity of Slc22a4 and Slc22a5 carnitine transporters were decreased in the spinal cord gray matter and motor neurons of SOD1(G93A) mice compared to the WT littermates A,B. Simi (n = 7) a. SLC22Apatients b. These patients .An MTT assay was performed on the ALS model of WT and MT cell lines following glutamate pretreatment to examine glutamate-induced cytotoxicity as well as the neuroprotective effect of LC and ALC under glutamate-induced cytotoxicity. Glutamate pre-incubation induced a significant decrease in cell viability compared to the control, but LC and ALC ameliorated glutamate-induced neuronal cell death in both NSC-34 cell lines A,B.3H]L-carnitine uptake and slc22a4 mRNA expression levels in both WT and MT cell lines. Pre-incubation with glutamate significantly increased the [3H]L-carnitine uptake rate, which was recovered of control level or decreased by pretreatment of LC and ALC in both NSC-34 cell lines (Similarly, the transport effects of LC and ALC pretreatment against glutamate excitotoxicity were examined by evaluating L-Carnitine uptake in NSC-34 cell lines was shown to be time-, temperature-, concentration-, ion-, energy-, and pH-dependent. The characteristics of its transport system were similar to those described in previous studies [3 and rotenone act by interfering with the electron transport chain in mitochondria and inhibition of the ATP synthase/production [3H]L-carnitine uptake was strongly inhibited in the presence of organic cationic compounds, including inhibitors as well as the substrates of the OCTN1 and 2 transporters, such as PLC, GBB, ALC, quinidine, verapamil, metformin, pyrilamine, TEA, clonidine, and DPH, in both NSC-34 cell lines [[ studies ,26,27. L studies  (Figure  studies ,17. As moduction ,26. Kineoduction . In addioduction . Furtherll lines ,32,33,34ll lines ,30,35. Nll lines .Amino acids, neurotransmitters and monocarboxylic acids as well as many other low-molecular-weight polar compounds can be transported to various cell lines using multiple transport systems that enable these substances to cross plasma membranes ,36. Simi+ and Cl−, which destabilizes intracellular Ca2+ homeostasis, thereby disrupting neuronal energy status and causing excitotoxic neuronal death, which constitutes the major root of neurodegenerative diseases, such as ALS [G93A mutant models [On the other hand, glutamate is an important neurotransmitter, its pretreatment overstimulates its receptors, leading to the activation of glutamate receptors and an influx of Nah as ALS . Additioh as ALS ,39. Howeh as ALS ,39. In oh as ALS ,41. As sh as ALS ,11,42. St models . These oOur results suggest that the energy-metabolizing compound carnitine can be transported to motor-neuron like cell lines using OCTN1 and OCTN2 transporters. LC transporter systems in the NSC-34 cell lines of mouse MT ALS models were altered compared to those of the WT control. Thus, LC and ALC show potential as good candidates for the prevention of neurodegenerative diseases, including ALS caused by the glutamate-induced motor neuronal cell death.


CCR1, CCR2, CCR3, and CCR5, the EBV DNA load and expression of EBV latent genes in nine EBV-carrying and four EBV-negative BL cell lines. We revealed that CCR1 is expressed at high mRNA and protein levels in two CD10-negative BL cell lines with co-expression of the EBV latent genes EBNA2, LMP1, and LMP2. Low levels of CCR2 transcripts were found in three BL cell lines. CCR3 and CCR5 transcripts were hardly detectable. Our data suggest that in vivo, CCR1 may be involved in the dissemination of BL cells and in the selection of BL cells with restricted EBV gene expression programs.Chemokines and their receptors regulate the migration of immune cells and the dissemination of cancer cells. CCR1, CCR2, CCR3, and CCR5 all belong to a single protein homology cluster and respond to the same inflammatory chemokines. We previously reported that CCR1 and CCR2B are induced upon Epstein-Barr virus (EBV) infection of B cells in vitro. EBV is present in almost all cases of endemic Burkitt lymphoma (BL); however, the contribution of EBV in the pathogenesis of the disease is not fully understood. Here, we analyzed the relation of the expression of  The chemokine-receptor network controls immune responses and directs the migration of immune cells. Chemokines transmit signals via chemokine receptors, which are G protein-coupled, cell-surface receptors. The C-C chemokine receptors, namely CCR1, CCR2, CCR3, and CCR5, belong to the same protein sequence homology cluster. They share responsiveness to multiple inflammatory chemokines. Inflammatory chemokines and their receptors are induced during inflammation and under many pathophysiological conditions. However, the roles of the chemokine receptors CCR1, CCR2, CCR3, and CCR5 in B lymphocytes remain unknown (reviewed in [CCR1 and CCR2B (an isoform of CCR2) mRNA and protein expression levels are upregulated in peripheral blood (PB) B cells upon EBV infection in vitro and in established lymphoblastoid cell lines (LCLs) [CCR1 or CCR2 nor their proteins were detected in the EBV-negative and EBV-carrying BL cell lines with latency I. Enhanced transcription of CCR2, CCR7, and CCR9 was previously detected in tonsillar B cells upon EBV infection in vitro [CCR2 starting from day 4 and after [In our previous work, we showed that s (LCLs) . We alsoin vitro . A recennd after .c-MYC oncogene is a major growth-promoting event. Endemic BL classically presents as a monoclonal cell tumor, in which every cell carries the c-MYC translocation and EBV. Most EBV-carrying BL tumors express only one EBV protein, EBNA1, from the alternative promoter Qp (a latency I type program) [EBV, a human B lymphotropic γ-1 herpesvirus, is associated with a number of human B cell lymphoproliferative disorders (LPDs), including BL, posttransplant lymphoproliferative disease (PTLD), lymphomas in immunodeficient patients, and diffuse large B cell lymphomas (DLBCLs). In actively proliferating PTLD and LCLs generated by a viral infection of resting B cells in vitro, EBV expresses the full set of latency type III (latency III) proteins: six nuclear antigens  and latent membrane proteins  . EBprogram) ,10,11 (rprogram) ,7,8). Deprogram)  and geneprogram)  indicateprogram) ,17,18,19program) ,20.CCR1, CCR2, CCR3, and CCR5 in BL cell lines with different EBV latency programs aiming to find a putative link between the expression of particular EBV latent genes and the expression levels of the chemokine receptors.Up to now, not much is known about the impact of EBV on eBL disease progression. To answer the question of whether the inflammatory chemokine receptors could be implicated in the selection of the EBV-infected BL cells in vivo, we analyzed the expression pattern of the known inflammatory chemokine receptors CCR1, CCR2, CCR3, and CCR5 in 13 BL cell lines, four EBV-negative cell lines , and in nine EBV-carrying cell lines. The EBV-carrying BL cell lines included three type I cell lines with latency I , four type II cell lines , and two type III cell lines (Mutu cl.99 and BL18). The Burkitt lymphoma cell lines used in the present study and references to the reported characteristics are shown in We analyzed the mRNA expression levels of the inflammatory chemokine receptors In the type II BL cell lines in our study, we did not detect LMP2A or LMP2B transcripts D. HoweveCCR2 mRNA expression levels we detected in three BL cell lines, with the following values normalized to TBP mRNA expression: 0.022 ± 0.001 in BL18; 0.040 ± 0.006 in Mutu cl.99; and 0.066 ± 0.002 in Jijoye P79. By comparison, in LCL-2mon cells, the value was 0.111 ± 0.031 (CCR2 (isoform CCR2B) was expressed and that the receptor was functional in LCLs and in three endemic BL cell lines , in which the EBV-encoded latent protein EBNA2 is abundantly expressed [Low  ± 0.031 A. We alsxpressed   or were hardly detectable in LCLs as in the type III and type II BL cell lines. The levels of the CCR5 normalized mRNA expression were as follows in the respective cell lines: 0.00082 ± 0.00003 in LCL-2mon; 0.00170 ± 0.00070 in LCL-1year; 0.00047 ± 0.00007 in BL18; 0.00161 ± 0.00001 in Mutu cl.99; 0.00228 ± 0.00054 in Jijoye P79; and 0.00006 ± 0.00000 in Akuba.In most BL cell lines in this study, CCR1 mRNA expression, with normalized expression values of 0.40 ± 0.07 and 0.46 ± 0.05, were detected in two CD10-negative type III BL cell lines, BL18 and Mutu cl.99 cells, respectively, in which the EBV latent genes EBNA2, LMP1, and LMP2A/2B were also expressed  [CCR1 mRNA expression levels and quantification of CCR1 transcript copy number were carried out using RT real-time PCR and primers targeting exons 1 and 2. The presence of the CCR1 ORF transcripts in the type III BL cell lines was confirmed using RT-duplex PCR  . Determiplex PCR G.Immunofluorescent staining definitively detected CCR1 protein in only two BL cell lines, Mutu cl.99 and BL18 cells, which is consistent with mRNA expression data E. The meExpression of the CCR1 protein (CD191) on the cell surface and percentages (%) of CD191-positive (CD191+) and CD10-positive (CD10+) cells were determined using flow cytometry; annexin V-stained (apoptotic) cells were excluded from the analysis. The percentages of CD191+ cells were assessed after recycling of the receptor to the cell surface, as a functional response to serum deprivation, after the cells were cultured in serum-free medium for 3 h . Serum dHowever, in Akuba cell line (the early passage type II BL cell line), only a small fraction of cells (~8.4%) carried CCR1. Cell surface expression of CCR1 was not detected in the CD10-positive and the EBV-negative , EBV-positive type I , or EBV-positive type II  BL cell lines . We did CCR1 or CCR2 . Howeiewed in ,19 and biewed in ,17. A priewed in . The autiewed in . Subsequpression .The majority of BL cell lines generated from tumors with EBV latency I maintained their original cell phenotype and latency I. However, some of the BL cell lines, upon the establishing, after 20–50 passages in culture, shifted to a lymphoblastoid phenotype and expressed EBV latency III proteins  ,11,20. FCCR1, CCR2, CCR3, and CCR5, in 13 BL cell lines with different expression patterns of the EBV latent genes EBNA2, LMP1, LMP2A, and LMP2B in comparison with LCLs. While only trace amounts of the CCR3 and CCR5 transcripts were found, and low levels of CCR2 mRNA expression were detected in type III BL cell lines and LCLs, CCR1 mRNA expression was high in all three LCLs and in all two type III BL cell lines. Neither CCR1 mRNA nor the CCR1 protein was detected in the EBV-negative and the latency I BL cell lines with the GC phenotype.In our previous work, we demonstrated that infection of PB B cells with EBV upregulated two inflammatory chemokine receptors, namely CCR1 and CCR2, and the expression of these chemokine receptors persisted in established LCLs . In the CCR1 and CCR2 mRNA expression levels as the transcript copy number per 1000 copies of the housekeeping gene TBP transcript (relative copy number). While the CCR1-transcript relative copy number in LCLs and two type III BL cell lines ranged from 969 in LCL-1year to 2670 in LCL-1mon and from 1080 in Mutu cl.99 to 1130 in BL18, the CCR2 mRNA expression levels, as in LCLs and in BL cell lines, were low compared to the CCR1. Specifically, we detected the CCR2 relative copy numbers of only 24, 50, and 26 in LCL-1year, LCL-2mon, and LCL-1mon, respectively, and of 10 in Mutu cl.99, and 23 in Jijoye P79 . ConseqConcluding, we demonstrated that only one CCR1 out of four known chemokine receptors in the family of inflammatory chemokine receptors was highly upregulated in the EBV-carrying LCLs and BL cell lines with co-expression of the EBV latent genes EBNA1, EBNA2, LMP1, and LMP2A/2B. In the CD10-positive BL cell lines, EBV-negative and EBV-positive with latency I program, none of the inflammatory chemokine receptors was expressed, which suggests that in vivo CD10-positive BL cells escape chemokine ligand-expressing immune cells, such as macrophages, T- and NK-lymphocytes. Our results may help to reveal mechanisms of endemic BL lymphomagenesis, contributing to understanding the selection of EBV-restricted latency BL-cell subsets within a tumor mass. We also speculate that in vivo endemic BL cells expressing CCR1 are prone to migrate toward ligand-expressing immune cells and ligand-rich blood organs, and therefore, in an immunocompromised state, dissemination of malignant cells may contribute to the pathogenesis of this cancer.The BL cell lines analyzed in this study along with the references to their origin, expression of EBV latent genes, and their reported characteristics are listed in CCR1, CCR2, CCR3, and CCR5 genes and the EBV genes, EBNA2, LMP1, LMP2A, LMP2B, and BZLF1, were assessed using real-time RT-PCR with the PerfeCTa SYBR Green FastMix  and the CFX96 Touch Real-Time PCR detection system . The mRNA expression levels of the genes of interest were normalized to the mRNA expression levels of the housekeeping gene TBP (encoding TATA box-binding protein). The presence of the CCR1 open reading frame (ORF) transcript was determined using RT-duplex PCR performed with two pairs of primers flanking the CCR1 ORF and matching the housekeeping gene GAPDH . We previously described the real-time RT-PCR procedure, the RT-duplex PCR protocol, and the corresponding primers in prior publications [The mRNA expression levels of the ications . The EBVications ; the EBVications ; and theications .CCR1 and TBP transcripts were determined by quantitative real-time RT-PCR. For copy number quantification, we applied standard curves based on serial dilutions of the plasmids with the inserted CCR1 or TBP transcripts derived from the established LCL-2mon reference line. The transcripts were cloned into the pCR-TOPO vector  and sequenced for verification. The EBV DNA copy number per cell for each cell line was quantified using the EBV Real-TM Quant kit .The copy numbers of the https://www.ncbi.nlm.nih.gov/igblast/, accessed on 1 January 2021).The analysis of the immunoglobulin heavy-chain variable region (IGHV) gene mutations was carried out according to the described protocol  using siWestern blot analyses were performed, using whole-cell lysates, with a mouse monoclonal anti-EBNA2 antibody  and a mouse monoclonal anti-LMP1 antibody . CCR1 immunostaining was performed using goat polyclonal anti-human CCR1 antibodies  and donkey anti-goat IgG, F(ab’)- fragment antibody conjugated with FITC . DNA in the cells, fixed with a mixture of methanol and acetone (1:1), was stained using Hoechst 33258 .For the three-color flow cytometry analysis, cells were stained with a mouse monoclonal anti-CD191 antibody  targeting the cell surface CCR1 or with a mouse monoclonal anti-CD192 antibody  targeting the cell surface CCR2 and a mouse monoclonal anti-CD10 , according to a standard protocol [


Breast cancer is a multifactor disease, and many drug combination therapies are applied for its treatment. Selenium derivatives represent a promising potential anti-breast cancer treatment. This study reports the cytotoxic activity of forty-one amides and phosphoramidates containing selenium against five cancer cell lines  and two nonmalignant cell lines (184B5 and BEAS-2B). MCF-7 cells were the most sensitive and the selenoamides I.1f and I.2f and the selenium phosphoramidate II.2d, with GI50 values ranging from 0.08 to 0.93 µM, were chosen for further studies. Additionally, radical scavenging activity for all the compounds was determined using DPPH and ABTS colorimetric assays. Phosphoramidates turned out to be inactive as radical scavengers. No correlation was observed for the antioxidant activity and the cytotoxic effect, except for compounds I.1e and I.2f, which showed dual antioxidant and antitumor activity. The type of programmed cell death and cell cycle arrest were determined, and the results provided evidence that I.1f and I.2f induced cell death via autophagy, while the derivative II.2d provoked apoptosis. In addition, Western blot analysis corroborated these mechanisms with an increase in Beclin1 and LC3-IIB and reduced SQSTM1/p62 levels for I.1f and I.2f, as well as an increase in BAX, p21 and p53 accompanied by a decrease in BCL-2 levels for derivative II.2d.  Cancer is considered a serious public health burden because it affects millions of people worldwide. Cancer is the second lethal disease globally, and about one out of six deaths are due to cancer . WorldwiIn this context, researchers worldwide have focused their attention on the synthesis of selenium-containing compounds due to their promising results against different types of cancer . SeveralN-acetylcysteine [In spite of the controversy about the use of antioxidants for cancer treatment, different clinical trials have demonstrated their efficacy . If we fcysteine , garlic cysteine  or flavocysteine  have beecysteine ,18. In tAccumulating evidence in the literature has illustrated that among the different selenated scaffolds, selenenocyanate ,20 and dIn this study, we provide an evaluation of the synthesized selenium derivatives as cytotoxic and antioxidant agents. The antiproliferative effect was tested in vitro using five human cancer cell lines as well as two nonmalignant cell lines. The antioxidant activity was determined by DPPH and ABTS assays. To better understand the potential mechanisms of action, the most active and selective compounds were further evaluated for their effect on the cell cycle distribution, cell death induction and alteration of different proteins related to autophagy or apoptosis processes.Synthesis, purification and characterization of the compounds have been previously described . Chemica2.Cell lines were provided by the European Collection of Cell Cultures (ECACC) or by the American Type Culture Collection (ATCC). Seven cell lines were used: MCF-7 (breast adenocarnicoma), 184B5 , CCRF-CEM (lymphoblastic leukemia), HT-29 (colon carcinoma), HTB-54 (lung carcinoma), BEAS-2B  and PC-3 (prostate carcinoma). MCF-7, CCRF-CEM, HT-29, HTB-54, BEAS-2B and PC-3 cell lines were grown in RPMI medium (Gibco) supplemented with 10% fetal bovine serum , 100 units/mL penicillin and 100 mg/mL streptomycin (Gibco). 184B5 cells were grown in DMEM/F12 medium supplemented with 5% FBS, 1 × ITS (Lonza), 100 nM hydrocortisone (Aldich), 2 mM sodium pyruvate (Lonza), 20 ng/mL EGF (Sigma-Aldrich), 0.3 nM trans-retinoic acid (Sigma-Aldrich), 100 units/mL penicillin and 100 mg/mL streptomycin. Cells were maintained at 37 °C and 5% COThe cytotoxic effect of each substance was tested by the MTT method. Each compound was initially dissolved in DMSO at a concentration of 0.01 M, and serial dilutions were prepared with nonsupplemented medium. The cytotoxic effect of each compound was tested at 50 and 10 µM as a first screening. Compounds with a cell growth percentage under 50% at 10 µM in at least one cell line were selected and tested at five different concentrations ranging between 0.01 and 100 µM.4 cells/well in 96-well plates were treated with increasing concentrations of the corresponding compounds for 48 h at 37 °C in a humidified atmosphere containing 5% CO2. Then, cells were incubated with 50 µL of MTT (2 mg/mL stock) for 4 h. The medium was then removed by aspiration, and the formazan crystals were dissolved in 150 mL of DMSO. Results are expressed as GI50, the concentration that reduces by 50% the growth of treated cells with respect to untreated controls (0.1% DMSO); TGI, the concentration that completely inhibits cell growth; and LC50, the concentration that kills 50% of the cells. Data were obtained from at least 3 independent experiments performed in quadruplicate. The standard error of the means (SEM) for the cytotoxic parameters was calculated applying the standard deviation formula to the mean values of each parameter for the three independent experiments performed.A total of 1 × 102 flasks  treated with DMSO (negative control), 6 µM of camptothecin (positive control) and different concentrations of I.1f, I.2f and II.2d.For the MCF-7 cell line, both cell cycle analysis and the cell death percentage were determined using the Apo-Direct kit (BD Pharmingen) based on the TUNEL technique, according to the manufacturer’s instructions. Cells were seeded in 25 cmAfter treatment, cells were collected and fixed with 1% paraformaldehyde (Sigma) in PBS (pH = 7.4), incubated in ice for 40 min, collected by centrifugation, washed with PBS and incubated with 70% ethanol for 30 min at −20 °C. After fixation, cells were washed twice with PBS and incubated for 1 h at 37 °C with FITC dUTP-DNA Labeling Solution. Cells were then rinsed and incubated in the dark for 30 min at room temperature with PI/RNase staining buffer before being analyzed by flow cytometry . Then, cells were treated with compounds I.1f (80 µM), I.2f (30 µM) and II.2d (5 µM) at different times (from 8 to 72 h) following the same methodology.For autophagy and caspase inhibition assays, cells were pretreated with 100 nM of autophagy inhibitor  and 50 µM of pan-caspase inhibitor  for 1 h. The cells were treated with the compounds I.2f (30 µM) and II.2d (20 µM) for 24 h and I.1f (80 µM) for 72 h. Samples were processed following the same methodology stated above. DMSO was used as a negative control for both inhibitors.g for 15 min at 4 °C to remove cell debris. The nonprotein fraction and supernatants were stored at −80 °C before use. Protein concentration was determined with the BCA Protein Assay. Cell samples (20–40 μg) were placed in SDS-sample buffer and 2% 2-β-mercaptoethanol, boiled for 5 min and subjected to SDS-PAGE on 12% Tris-glycine gels. Separated proteins were transferred onto 0.22 μm nitrocellulose membranes at 100V for 1 h. The membranes were incubated in blocking solution (5% nonfat dry milk-TBS-Tween-20) for 1 h at room temperature. Primary specific antibodies were incubated in 5% milk-TBS-Tween-20  to detect LC3B, Beclin-1 (D40C5), SQSTM1/p62, AMPK, JNK, BCL-2, BAX, p-53 and p21  and actin (Santa Cruz Biotechnology). After incubation with the HRP-conjugated secondary antibody  in 5% milk-TBS-Tween-20 , a chemiluminescence kit was used for visualization.After treatment, cells were lysed in cell lysis buffer consisting of: 20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1% Triton X-100, supplemented with protease inhibitors and phosphatase inhibitors (10 mmol/L sodium fluoride and 10 mmol/L sodium orthovanadate) for 30 min on ice. Lysates were centrifuged at 13,200× control refers to the absorbance of the negative control, and Asample refers to the absorbance of the tested compounds. Results are expressed as a percentage of DPPH radical scavenged ± SEM. The DPPH  assay measures the hydrogen donation ability of the antioxidant to convert the stable DPPH free radical into 1,1-diphenyl-2--hydrazine. After radical reaction with the compounds, a decrease in the absorbance was detected at 517 nm, which is accompanied by a change of color from violet to light-yellow. The target compounds were dissolved in methanol to make 1 mg/mL stock solutions, which were diluted to five concentrations as test samples. Ascorbic acid was used as a standard, and the experiment was carried out on 96-well plates. The DPPH solution was prepared at a 100 µM concentration. For each test, 100 µL of DPPH solution was added to 100 µL of methanolic solution containing the tested derivatives, and the absorbance was determined at different time points. All the measurements were carried out in triplicate. Results were expressed as the percentage of the radical scavenged, calculated using the following formula:•+ with potassium persulfate . This cocktail was kept overnight away from light at room temperature. Then, this ABTS•+ reaction mixture was diluted with a 50% ethanolic solution in order to achieve absorbance values of 0.70 ± 0.02 at 734 nm for measurements. Finally, 1 mg/mL stock solutions for the selected derivatives were formed in absolute ethanol and 20 µL of these solutions were added to 180 µL of the diluted ABTS•+ cocktail. After 6 min of incubation, absorbances at 734 nm were registered. A 50% ethanolic solution was used as blank along with Trolox (TROL) and ascorbic acid (Asc) as positive controls. All of the determinations were performed in triplicate using 96-well plates. The same time intervals as in the DPPH assay were also measured. The ability to scavenge ABTS•+ was calculated using the following formula:control refers to the absorbance of the negative control and Asample refers to the absorbance of the tested compounds. The results are expressed as percentage of ABTS radical scavenging ± SEM.ABTS radical scavenging was additionally assayed with a colorimetric assay following a previous methodology . Brieflyt-test. * Statistical significance was defined as p < 0.05 (*); p < 0.01 (**) or p < 0.001 (***).Data were expressed as the mean ± SEM and analyzed by the Student’s The route adopted for the synthesis of the novel forty-one compounds presented in this work has been previously reported ,31. StruThe synthesis of both series of compounds followed similar synthetic routes. Briefly, 4-aminophenylselenocyanate (0A) and bis-(4-aminophenyl)diselenide (0B) were achieved as previously reported . For serFor series II, 0A and 0B were treated dropwise with the corresponding phosphoryl chlorides under different temperatures, atmospheric conditions and reaction times .Herein, two series of compounds based on amides (series I) and phosphoramidates (series II) derived from 4-aminophenylselenocyanate and bis-(4-aminophenyldiselenide) were tested in vitro against cell lines derived from breast adenocarcinoma (MCF-7), lymphoblastic leukemia (CCRF-CEM) and colon adenocarcinoma (HT-29). Evaluation was performed at 48 h of treatment following the MTT -2,5-diphenyltetrazolium bromide) methodology as previously described . The in 50, TGI and LC50 values were calculated from the curves and are shown in 50 (184B5)/GI50 (MCF-7) and GI50 (BEAS-2B)/GI50 (HTB-54) . The presence of electron-withdrawing groups  in the phenyl ring favored the cytotoxic activity in the amides, but in the phosphoramidates, the best results were obtained for aliphatic chains methoxy  and ethoxy . Additionally, the replacement of oxygen  by sulfur  resulted in the abrogation of the activity. In relation to selenium entity, amides with selenocyanate motif were more active than the corresponding diselenides, and this is particularly evident in PC-3 . However, no significant differences were observed for phosphoramidates. Twelve compounds: 1b, 2b, 1e, 2e, 1f, 2f, 1h and 2h (series I) and compounds 1b, 2b, 1d and 2d (series II) showed cell growth below 50% at a 10 µM concentration. Hence, they were tested at five concentrations between 0.01 and 100 µM against five cancer cell lines, MCF-7, CCRF-CEM, HT-29, HTB-54 (lung carcinoma) and PC-3 (prostatic adenocarcinoma), and two nonmalignant cells, 184B5 and BEAS-2B. GI(HTB-54) . CisplatThe lack of selectivity toward cancerous cells is one of the major issues during the discovery and development of new anticancer agents. Therefore, we evaluated the cytotoxic activity of our compounds against two cell lines derived from nonmalignant breast tissue (184B5) and no malignant bronchial epithelium (BEAS-2B) . Data anCollectively, considering all the results, compounds I.1f, I.2f and II.2d showed the most significant cytotoxicity against all the screened cell lines along with acceptable safety and were picked for further pharmacological research in the breast adenocarcinoma cell line MCF-7.In a first approach, the radical scavenging activity for all the synthesized compounds was characterized by the DPPH colorimetric assay at a dose of 0.03 mg/mL of each compound and six different time points . Ascorbic acid and Trolox were used as radical scavenger gold standards. Three diselenides  and three selenocyanates  from series I showed DPPH activity inhibition values greater than 35% after 30 min A of treaIn a second approach, the antioxidant activity of the six compounds that showed DPPH activity inhibition was also further confirmed using the ABTS colorimetric assay C. SurpriApoptosis and cell cycle arrest are typical mechanisms of action for many anticancer drugs, including selenocompounds ,35,36. IAs seen in To further understand the mechanism of action of these potent compounds, their effects on cell cycle distribution in MCF-7 were studied by flow cytometry. This assay was carried out with different concentrations  and diffAfter treatment with I.1f, I.2f and II.2d at various time points (24–72 h for I.1f and 8–48 h for I.2f and II2d), the results suggested that these compounds arrested cell cycle in a time-dependent fashion .Apoptosis and autophagy are considered as two recognized pathways for anticancer agents due to their effects on cell survival ,38. In tAs illustrated in Conversely, preincubation with wortmannin A could nTo further confirm the programmed cell death pathway, the expression levels of several markers of autophagy  and apopBax is an important proapoptotic protein implicated in apoptosis induction. On the contrary, Bcl-2 is an important antiapoptotic protein that suppresses apoptosis. The balance between Bax and Bcl-2 is very important for judgmental cell apoptosis . The exp50 values < 1 µM . These compounds were approximately three-fold more potent than cisplatin, a reference drug used clinically. Additionally, in vitro radical scavenging studies have shown that only series I compounds (amide derivatives) present antioxidant activity. Surprisingly, no phosphoramidate showed this effect. No correlation was found for either structure- or cytotoxicity-antioxidant activity, except for compound I.2f. Moreover, compounds I.1f, I.2f and II.2d showed the highest SI values when comparing the cytotoxic activity against 184B5 nonmalignant cell lines and were selected for further biological studies. Flow cytometry studies suggested that amides I.1f and I.2f induced cell cycle arrest at the S phase and cell death in a time- and dose-dependent manner and cell death through an autophagy process. Meanwhile, phosphoramidate II.2d flow cytometric studies showed an early caspase-dependent cell death at low doses and cell cycle arrest at G0/G1 phase at a 5 µM dose. However, as the dose increases, the number of subdiploid cells in the SubG1 phase also increases significantly. Both autophagy processes caused by I.1f and I.2f and apoptosis induction caused by II.2d were confirmed by Western blot analysis.Forty-one new amides and phosphoramidates bearing selenocyanate and diselenide scaffolds were prescreened against three tumor cell lines at two doses. Twelve compounds were selected and tested against a panel of five tumor cell lines and two nonmalignant cell lines. The breast tumor cell line MCF-7 was the most sensitive, with six compounds showing GI


DR4 and DR5 genes may be involved in resistance of leukemia cells to immunotherapies due to TRAIL-resistance. We analyzed the DR4 and DR5 methylation status in 32 BCP-ALL cell lines by sequencing their bisulfite PCR products with a next-generation sequencer. The DR4 and DR5 methylation status was significantly associated with the gene and cell-surface expression levels and the TRAIL-sensitivities. In the clinical samples at diagnosis (459 cases in the NOPHO study), both DR4 and DR5 genes were unmethylated in the majority of cases, whereas methylated in several cases with dic, MLL-rearrangement, and hypodiploidy, suggesting that evaluation of methylation status of the DR4 and DR5 genes might be clinically informative to predict efficacy of immunotherapy in certain cases with such unfavorable karyotypes. These observations provide an epigenetic rational for clinical efficacy of immunotherapy in the vast majority of BCP-ALL cases.Immunotherapies specific for B-cell precursor acute lymphoblastic leukemia (BCP-ALL), such as anti-CD19 chimeric antigen receptor (CAR) T-cells and blinatumomab, have dramatically improved the therapeutic outcome in refractory cases. In the anti-leukemic activity of those immunotherapies, TNF-related apoptosis-inducing ligand (TRAIL) on cytotoxic T-cells plays an essential role by inducing apoptosis of the target leukemia cells through its death receptors (DR4 and DR5). Since there are CpG islands in the promoter regions, hypermethylation of the  BCR-ABL1-positive leukemia cells, which are clinically sensitive to the GVL effect [MLL (KMT2A)-rearranged (MLLr) acute lymphoblastic leukemia (ALL) cells, which are clinically resistant to the GVL effect [Two recently established immunotherapies specific for B-cell precursor acute lymphoblastic leukemia (BCP-ALL) have dramatically improved the therapeutic outcome in poor prognostic BCP-ALL patients. One is chimeric antigen receptor (CAR) T-cell therapy targeting at CD19 . Both thL effect  frequentL effect . In contL effect , generalL effect . We alsoL effect . MoreoveL effect . AccordiDR4 [DR4 and/or DR5 genes may be associated with the DR4 and DR5 expression and consequently with the TRAIL sensitivity in BCP-ALL cells. However, to date, little is known about the relevance of the epigenetic modification of the DR4 and DR5 genes in BCP-ALL.Hypermethylation of the CpG island in the gene promoter is an epigenetic modification of gene expression. We previously reported that cell lines and patients’ samples of T-cell ALL (T-ALL) showed a TRAIL resistance in association with low cell-surface expression levels of DR4 and DR5 . Semi-quDR4 . These oDR4 and DR5 gene promoter in BCP-ALL cell lines by sequencing their bisulfite PCR products with a next-generation sequencer (NGS). We found that hypermethylation of the DR4 and DR5 CpG islands is highly associated with a lack of cell-surface expression of DR4 and DR5, and TRAIL resistance in BCP-ALL cell lines. We further investigated the significance of the methylation status of the DR4 and DR5 genes in clinical samples of BCP-ALL patients.In the present study, we quantified the methylation status of the CpG islands of the MLLr-ALL cell lines  [BCR-ABL1-positive ALL cell lines  [TCF3-PBX1-positive ALL cell lines ; four TCF3-HLF-positive ALL cell lines  [ETV6-RUNX1-positive ALL cell lines (KOPN79 and Reh); two MEF2Dr-ALL cell lines (KOPN61 and KOPN70); and two DUX4r-ALL cell lines  were used in this study (2 at 37 °C. Forty-nine cryopreserved samples of childhood BCP-ALL (35 samples at diagnosis and 14 samples at relapse after chemotherapy) (Nine  KOCL69) ; six BCR YAMN91) ; seven T HAL-O1) ; and twois study . All celtherapy)  were ana4 cells/well) were cultured in the absence or presence of 100 ng/mL of rhsTRAIL in triplicate in 200 μL of RPMI1640 medium supplemented with 10% FCS in a flat-bottomed 96-well plate. The plates were incubated for 42 h, pulsed for the last 6 hours of the incubation with 3H-thymidine (1 μCi/well) and harvested onto glass-fiber filters. The level of radioactivity incorporated into DNA was measured by liquid scintillation counting. The percent inhibition by rhsTRAIL was calculated as the ratio of the radioactivity to that in the absence of rhsTRAIL.Sensitivities to TRAIL were determined as previously reported ,15,18,191 or mAb on ice for 30 min. After washing, the cells were incubated with phycoerythrin-conjugated streptavidin  on ice for 30 min, and then analyzed by flow cytometry. The relative florescence intensity (RFI) was calculated as the ratio of the mean fluorescence intensity of specific staining to that of control staining.Cell-surface expression of DR4 and DR5 was determined as previously reported using the monoclonal antibodies (mAbs) specific to DR4 and DR5 ,15,18,19DR4 and DR5 were quantified as previously reported by real-time reverse transcription polymerase chain reaction (RT-PCR) analysis [DR4 and DR5 transcripts, triplicated samples containing 9 μL of cDNA with 10 μL of Taqman Universal PCR Master Mix  and 1 μL of 20 × Assays-on-demand Gene Expression Product  were pre-incubated at 50 °C for 2 min and subsequently at 95 °C for 10 min. Amplification was obtained by 40 cycles of reaction at 95 °C for 15 sec and 60 °C for 1 min. Fluorescence data were quantitatively analyzed on ABI Prism 7500 Sequence Detection System (Applied Biosystems). Nalm1, a TRAIL sensitive chronic myelogenous leukemia blast crisis -derived cell line that expresses DR4 and DR5 [GAPDH; Hs 99999905_m1, Applied Biosystems) was performed.Gene expression levels of analysis ,19. Tota and DR5 , was useDR4 gene with forward primer (5′-GGAAGGAAGTTTAGGGTTAGTTAATAG-3′) and reverse primer (5′-TACCAAATCAATCCAAAAAACAAC-3′) and DR5 gene with forward primer (5′-AAGTGTTTTTTTTAATTTATTTTTTTTAAG-3′) and reverse primer (5′-CAACTACAAATTCCACCACAAATTA-3′) using one cycle of 95 °C for 4 min, 40 cycles of 95 °C for 30 sec, 55 °C for 30 sec, 72 °C for 1 min, with a final cycle of 72 °C for 7 min. For conventional Sanger sequencing, PCR products were cloned into a pTAC-2 vector using a TA PCR Cloning Kit  and sequenced. For next-generation sequencing, amplicon libraries were generated by Ion Plus Fragment Library Kit  and Ion Xpress Barcode Adaptors Kit (Thermo Fisher Scientific). Briefly, 50 ng amplicon was end-repaired, nick-repaired, and Ion Torrent adapters P1 and Barcode were ligated with DNA ligase. Following Agencourt AMPure XP purification , the individual libraries were quantified by quantitative real-time PCR then diluted, and finally pooled in equimolar ratios. The libraries were processed with the Ion OneTouch™ 2 System using Ion PGM™ Template OT2 400 Kit (Thermo Fisher Scientific) to produce 400 base-read libraries. Sequencing was performed by an Ion PGM™ Hi-Q Sequencing Kit (Thermo Fisher Scientific) using 850 flows on the Ion 318 Chip Kit v2 (Thermo Fisher Scientific) according to the manufacturer’s protocol. After sequencing, single processing and base-calling were performed using Torrent Suite 5.0.2 (Thermo Fisher Scientific). Methylation analysis was performed using MethylationAnalysis_Amplicon plug-in v1.3 (Thermo Fisher Scientific).Bisulfite PCR was performed as previously reported . GenomicETV6-RUNX1, TCF3-PBX1, iAMP21, dic, BCR-ABL1, MLLr, hypodiploid (<45 chromosomes), and polyploid (>67 chromosomes). The data of the paired samples at diagnosis and at relapse were also analyzed in 24 relapsed BCP-ALL cases. Percent methylation of DR4 and DR5 was evaluated by mean methylation level of CG dinucleotides that were annotated as both 0–200 bases upstream of the transcriptional start site (TSS200) and CpG island using Illumina HumanMethylation450 BeadChip  [Gene methylation of childhood BCP-ALL clinical samples were investigated using 450 k DNA methylation array database of Nordic Pediatric Hematology and Oncology (NOPHO)  . Among 7Mann–Whitney U test, Fisher’s exact test, Chi-square test, and Spearman’s correlation analysis were performed using R software version 3.5.2 (R Core Team 2018).DR4  was observed between the mean percent methylation of the DR4 gene and that of the DR5 gene in 32 cell lines  in all of nine MLLr-ALL and two MEF2Dr-ALL cell lines, while it was unmethylated (<1%) in all of four TCF3-HLF-positive ALL cell lines (DR5 gene (DR5 gene was methylated (>10%) in six of nine MLLr-ALL cell lines and all of two MEF2Dr-ALL cell lines, while it was unmethylated in all of four TCF3-HLF-positive ALL and two ETV6-RUNX1-positive ALL cell lines. Despite being limited to a small number of cell lines, these observations suggest that methylation status of the DR4 and DR5 genes may be associated with karyotypes of BCP-ALL cell lines.We next analyzed an association of the methylation status of the ll lines G. A simiDR5 gene H. The DRDR4 and DR5 genes and the gene and cell-surface expression levels of DR4 and DR5 in 32 BCP-ALL cell lines. Mean percent methylation of DR4 gene showed a significant negative correlation, both with the gene expression level of DR4 (R2 = 0.37) and with the cell-surface expression level of DR4 (R2 = 0.39)  and the cell-surface expression level of DR5 (R2 = 0.38)  A. Simila = 0.38) B.DR4 and DR5 genes with the sensitivity to anti-leukemic activity of rhsTRAIL [DR4 gene showed a significant negative correlation with the percent inhibition by rhsTRAIL (R2 = 0.58)  between mean percent methylation of the DR5 gene and percent inhibition by rhsTRAIL was 0.23   as well as six unmethylated cell lines (DR4 and DR5 <1%)  were highly sensitive to rhsTRAIL (percent inhibition ≥80%). In contrast, three of five DR5-preferentially unmethylated cell lines  as well as 13 of 18 methylated cell lines (DR4 and DR5 ≥1%) were resistant to rhsTRAIL (percent inhibition <25%). Among them, all of the 11 highly methylated cell lines (DR4 and DR5 ≥10%) were resistant to rhsTRAIL (percent inhibition <25%). These observations indicate that the methylation status of DR4 and DR5 (particularly DR4) is tightly associated with rhsTRAIL sensitivity in BCP-ALL cell lines  C. To comwas 0.23 C. Since sitivity D. Of notll lines E.DR4 and DR5 genes in 49 childhood BCP-ALL samples by sequencing bisulfite PCR-product using the NGS. In contrast to cell lines, mean percent methylation levels of the DR4 and DR5 genes were less than 1% in the majority of the samples  was observed between the mean percent methylation of the DR4 gene and that of the DR5 gene in 49 samples  [R2 = 0.35, p < 0.001) was observed between the percent methylation of the DR4 gene and that of the DR5 gene in 459 BCP-ALL samples -positive; 2 of 28 MLLr-positive; 1 of 5 hypodiploid) ALL samples  ALL samples  in all of 187 high hyperdiploid ALL, 164 ETV6-RUNX1-positive ALL, 10 iAMP21-positive ALL, and 19 BCR-ABL1-positive ALL samples. In the bone marrow samples in complete remission and normal T-cells and B-cells, % methylation of the DR4 and DR5 genes were approximately 2–3%. In the majority of samples from patient at relapse, the percent methylation of DR4 and DR5 genes was almost unchanged compared to that of diagnosis. However, in the paired samples at diagnosis and at relapse, methylation levels of DR4 and DR5 genes were upregulated in several cases at relapse  . A signi samples C. In com samples D and 16  samples E showed  relapse F. These DR4 and DR5 genes by using a BCP-ALL cell line as a model system. In BCP-ALL cell lines, the methylation status of the DR4 and DR5 genes was associated with their mRNA and cell-surface expression levels and their rhsTRAIL sensitivities, indicating that epigenetic modification of the DR4 and DR5 genes due to hypermethylation is a mechanism for TRAIL resistance in BCP-ALL. However, we observed discrepancies in the methylation status of the DR4 and DR5 genes between the cell lines and the clinical samples. The DR4 and the DR5 genes were not methylated in the majority of clinical samples. This difference in methylation status may be partly attributed to the different distribution of karyotypes between the cell lines and clinical samples. In BCP-ALL cell lines, the DR4 and DR5 genes were unmethylated in ETV6-RUNX1-positive ALL cell lines, while being frequently methylated in MLLr-ALL cell lines. Similarly, in the genome-wide DNA methylation data of BCP-ALL samples in the NOPHO cohort, the DR4 and DR5 genes were exclusively unmethylated in the samples with ETV6-RUNX1 but relatively frequently methylated in the samples with MLLr. In our series of BCP-ALL cell lines, only 6% (2/32) of cell lines had ETV6-RUNX1, while 31% (10/32) of cell lines showed the MLLr karyotype. In contrast, in the NOPHO cohort, over one-third (164/459) of the samples had ETV6-RUNX1, while only 6% (28/459) of the samples showed the MLLr karyotype. We also observed upregulation of the DR4/DR5 methylation status in several relapsed cases  cell lines and is moderately sensitive to rhsTRAIL , suggesting that the DR5 gene, but not the DR4 gene, plays an essential role in CAR T-cell cytotoxicity against Nalm6 due to unmethylated status of the DR5 gene. Meanwhile, we previously reported that cell-surface expression of TRAIL on anti-CD19 CAR T-cells is upregulated by the co-culture with targeted BCP-ALL cells [As a new therapeutic modality, immunotherapy using anti-CD19 CAR T-cells and blinatumomab is promising for refractory BCP-ALL cases. In a recent genome-wide CRISPR-Cas9 screen of Nalm6, a BCP-ALL cell line, toxicity . In our LL cells . These oDR4 and DR5 genes is associated with rhsTRAIL resistance of BCP-ALL cell lines. However, gene silencing of the DR4 and DR5 genes due to hypermethylation was uncommon in the majority of BCP-ALL cases at diagnosis. Accordingly, resistance to immunotherapy due to TRAIL resistance as a result of hypermethylation of the DR4 and DR5 genes is unlikely in the majority of BCP-ALL cases, at least at disease onset. In several relapsed cases of the NOPHO cohort study, upregulation was observed in the methylation status of the DR4 and DR5 genes. Although the precise treatment in these cases was unknown, this observation suggests the possibility that acquired hypermethylation of the DR4 and/or DR5 genes may be one of mechanisms for relapse particularly after immunotherapy.Blinatumomab is a bispecific T-cell engager antibody simultaneously binding CTLs and CD19-positive BCP-ALL cells , suggestDR4 and/or DR5 genes was observed in certain karyotypes such as dic, MLLr, and hypodiploidy. Thus, when the BCP-ALL patients with these karyotypes are treated by immunotherapy, methylation status and/or gene/cell-surface expression levels of the DR4 and DR5 genes might be useful biomarkers to predict therapeutic responses. In this context, we previously reported that TCF3-HLF-positive ALL cells are highly sensitive to TRAIL, since TCF3-HLF fusion transcription factor effectively transactivates the DR4 and DR5 gene expression [DR4 and DR5 genes are unmethylated in all of four TCF3-HLF-positive ALL cell lines. TCF3-HLF-positive ALL is the most unfavorable type of childhood BCP-ALL due to resistance to conventional chemotherapy [TCF3-HLF-positive ALL patients resistant to a conventional chemotherapy [TCF3-HLF-positive ALL patients.Of importance, in the analysis of clinical samples, hypermethylation of the pression . Indeed,otherapy ,26. Of notherapy . Our finDR4 and DR5 genes is unlikely in the majority of BCP-ALL cases, particularly in the cases with favorable karyotypes such as hyperdiploidy and ETV6-RUNX1. Since TRAIL/death receptor system plays an essential role in the anti-leukemic activities of immunotherapy using anti-CD19 CAR T-cells, our findings provide an epigenetic rationale for clinical efficacy of immunotherapy in BCP-ALL patients. Moreover, in certain BCP-ALL cases with unfavorable karyotypes such as dic , MLLr, and hypodiploidy, evaluation of methylation status of the DR4 and DR5 genes might be clinically informative to predict the efficacy of immunotherapy.The present study revealed that TRAIL-resistance due to hypermethylation of the


Metastases formation involves the formation, circulation and seeding of cohesive group of tumor cells called circulating tumors cells clusters at distant organs from the primary tumor. These clusters have a much higher metastatic potential than individual circulating tumor cell, it is therefore important to understand the molecular mechanisms involved in their formation. To this aim, in this study, from the analysis of the relationship between in vitro aggregation quantitative characterization of 25 cancer cell lines and their expression data, we identified genes significantly associated with aggregation. Interestingly, we found that these genes were strongly correlated with the transcriptional signature induced by HDAC inhibitors treatment and we finally showed experimentally that two HDAC inhibitors inhibits tumor cells cluster formation in vitro. These results open new therapeutic perspectives to prevent metastasis formation.Characterization of the molecular mechanisms involved in tumor cell clustering could open the way to new therapeutic strategies. Towards this aim, we used an in vitro quantitative procedure to monitor the anchorage-independent cell aggregation kinetics in a panel of 25 cancer cell lines. The analysis of the relationship between selected aggregation dynamic parameters and the gene expression data for these cell lines from the CCLE database allowed identifying genes with expression significantly associated with aggregation parameter variations. Comparison of these transcripts with the perturbagen signatures from the Connectivity Map resource highlighted that they were strongly correlated with the transcriptional signature of most histone deacetylase (HDAC) inhibitors. Experimental evaluation of two HDAC inhibitors (SAHA and ISOX) showed that they inhibited the initial step of in vitro tumor cell aggregation. This validates our findings and reinforces the potential interest of HDCA inhibitors to prevent metastasis spreading. The detection and monitoring of circulating tumor cells (CTC) have been the subject of major interest for several years to study and understand cancer pathology and to monitor the therapeutic response ,2. IndeeCell–cell adhesion involves multiprotein complexes located in intercellular junctions. In vertebrates, these cellular structures mostly comprise adherens junctions, gap junctions, desmosomes, and tight junctions. Proteins of the cadherin family are involved in adherens junctions (E-cadherin) and desmosomes (desmoglein and desmocolin); occludin, claudin, and ZO proteins are the most important components of tight junctions and Gap junctions connect two adjacent cells by establishing the continuity of connexons formed by connexins.Several years ago, it was shown that increased expression of E-cadherin, a major component of cell adherens junctions, enhances the formation of cell aggregates . This isTo identify additional molecular actors involved in the formation and/or stability of cancer cell clusters, study of the modalities and regulation of clustering also relies on in vitro assays to monitor intercellular interactions. We previously developed such an assay that we used to demonstrate the key positive role of E-cadherin and of two desmosomal proteins (DSG2 and DSC2), which interact with plakoglobin at this junction, in cancer cell aggregation . We alsoThe characterization of the molecular mechanisms involved in tumor cell clustering could open the way to new therapeutic perspectives. Therefore, it is essential to identify the regulators of this process and their specific roles. With this aim in mind, we chose an approach based on a basic observation. Specifically, among tumor cell lines of epithelial origin, some display very high anchorage-independent clustering capacities in vitro and aggregate very rapidly, while others have very limited, if any, clustering capacity. Therefore, we analyzed the anchorage-independent aggregation capacity of 25 tumor epithelial cell lines. Then, we selected three quantitative aggregation dynamic parameters to evaluate their relationship with gene expression data for these cell lines extracted from the CCLE database . This apAltogether, our observations support and strengthen the idea that deciphering the mechanism of tumor cell clustering and identifying the underlying regulatory pathways and interfering compounds might lead to new therapeutic opportunities.2 at 37 °C in DMEM , without or with 20 IU/L insulin , or in RPMI1640  medium according to the supplier’s recommendations, both supplemented with 10% fetal calf serum  and 1% penicillin/streptomycin . To study the impact of SAHA and ISOX, cells were incubated in culture medium containing 5 µM ISOX for 8 h or 10 µM SAHA for 24 h, then trypsinized and seeded for aggregation assay in culture medium containing the same concentration of ISOX and SAHA.The 25 cancer cell lines  were cul®, Corning, New York, NY, USA), except in the 36 peripheral wells to avoid edge effects. Plates were centrifuged at 400 g for 4 min, and then cell aggregation in each well was followed by time-lapse video-microscopy.The cell clustering assay used in this study was adapted with slight modifications from the one described in our previous reports ,13. SpecBased on our previous study ,13, imagFor each position/well/time, the whole stack of 2D images is fused into one single in-focus image. This process is based on a Laplacian of Gaussian filter to detect the in-focus image regions, followed by a Gaussian blending of these regions taken from the different focal planes.The background signal of the resulting in-focus image is estimated using a morphological opening and then subtracted.A Gaussian smoothing filter is then applied and the image intensities adjusted , followed by a simple automated thresholding, resulting in a binary mask that corresponds to all cell aggregates.2, because the XY resolution is 0.645 µm at 10× magnification). Detected objects with an area smaller than this threshold are excluded from the analysis. Holes inside the binary objects are excluded in the two object categories to measure standard parameters: number of aggregates, normalized area to the initial time point, and circularity. This image analysis pipeline was developed within a parallelized processing architecture using multi-core processors.Cell aggregates are individually detected in the binary image as connected components that match several criteria, and are divided in two groups according to their area using a threshold of 10,000 pixels ; a circularity of 1 indicates a perfect circle . [The gene expression data for the 25 cell lines used in this study were downloaded from the CCLE website e 2019). ,17. A spe 2019).  was used. ,17. A e 2019). .https://clue.io/connectopedia (accessed on: 31 August 2020).The Connectivity Map (CMap) is a resource that uses cell responses to perturbation to find relationships between diseases, genes, and therapeutics. The CMap database contains over one million gene expression signatures upon treatment of a variety of cell types with many different perturbagens . This resource has been successfully used in various studies in the field of cancer research to identify new candidates based on original signatures and to propose new drug repurposing opportunities  behaved very similarly in this assay.Our aim was to analyze the relationship between this variability and the gene expression profiles of the tested cell lines to identify new regulators of tumor cell clustering. Thus, to compare all 25 cell lines, we extracted three quantitative parameters that precisely described the aggregation kinetics and characteristics: the area-2h (early aggregation dynamics), the AUC (whole aggregation process), and circularity. These values are presented in www.broadinstitute.org/ccle accessed on: 4 June 2019), a collaboration between the Broad Institute and Novartis [To identify genes the expression of which was associated with the aggregation quantitative parameters, we extracted the expression data of 19,851 genes for the 25 cell lines used in our study from the Cancer Cell Line Encyclopedia (CCLE) project . The complete list of the median tau scores for the perturbagens is provided as As the genes associated with area-2h and AUC were different from those associated with circularity, we could not identify putative regulators of tumor cell aggregation directly with the sparse PLS method. Therefore, we used the CMap resource  to compaThese results suggest that HDAC inhibitors could regulate the capacity of cancer cells to form clusters.These results and a recent publication showing that HDAC inhibitors reduce CTC cluster size  led us tp < 0.0001) and the AUC  were significantly higher in SAHA-treated cells than controls. We obtained similar results in cells incubated with ISOX , which confirms the reliability of the methodology used.We focused on three quantitative parameters: area-2h, AUC, and circularity. First, we identified genes that showed expression variations in the 25 cell lines associated with these aggregation parameters using data from the CCLE transcriptional database ,17. ThenA recent comparative analysis  demonstr


Further in vitro and in vivo analyses in tumor-bearing mice are ongoing in our lab to support this claim and to unravel the full therapeutic efficacies of NSC777205 and NSC777207 in CRC.Genetic and environmental factors play important roles in cancer progression, metastasis, and drug resistance. Herein, we used a multiomics data analysis to evaluate the predictive and prognostic roles of genetic and epigenetic modulation of c-MET (hepatocyte growth factor receptor)/epidermal growth factor receptor (EGFR) in colorectal cancer (CRC). First, we found that overexpressions of c-MET/EGFR were associated with the infiltration of tumor immune cells and cancer-associated fibroblasts, and were of prognostic relevance in CRC cohorts. We also observed that genetic alterations of c-MET/EGFR in CRC co-occurred with other gene alterations and were associated with overexpression of messenger (m)RNA of some cancer hallmark proteins. More specifically, DNA-methylation and somatic copy number alterations of c-MET/EGFR were associated with immune infiltration, dysfunctional T-cell phenotypes, and poor prognoses of the cohorts. Moreover, we describe two novel gefitinib-inspired small molecules derivatives of 3-phenyl-2H-benzo[e] -oxazine-2,4(3H)-dione, NSC777205 and NSC777207, which exhibited wide-spectrum antiproliferative activities and selective cytotoxic preference for drug-sensitive and multidrug-resistant melanoma, renal, central nervous system, colon, and non-small cell lung cancer cell lines. We further provided in silico mechanistic evidence implicating c-MET/EGFR/phosphatidylinositol 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibition in anticancer activities of those compounds. Our overall structure-activity relationship study revealed that the addition of an –OCH According to 2020 global cancer statistics, with more than 1.9 million new cases and 935,000 deaths, colorectal cancer (CRC) ranked third in terms of incidence, but second in terms of mortality in 2020 . CRC is Both genetic and environmental factors play important roles in the etiology of CRC. To date, several molecular hallmarks have been associated with CRC . These hMET gene, while the EGFR belongs to the ErbB family of RTKs, and is a trans-membrane protein. The binding of ligands to the extracellular domain of these receptors induces the formation of receptor homo- or heterodimers and subsequent activation of the intrinsic tyrosine kinase domain , is a tyrosine kinase receptor (RTK) that in humans is encoded by the e domain , which fe domain .in silico mechanistic evidence implicating c-MET/EGFR/phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibition in anticancer activities of these compounds.Several treatment modalities exist, including immunotherapy, radiotherapy, neoadjuvant and palliative chemotherapies, laparoscopic surgery for primary disease, and more-aggressive resection of metastatic disease, and these provide alternatives for patients with primary and metastatic CRC . Howeverhttp://www.swissadme.ch) (http://way2drug.com/dr) (https://www.cbligand.org/BBB/) which operates based on support vector machine (SVM) and LiCABEDS algorithms on four types of fingerprints of 1,593 reported compounds (http://www.way2drug.com/PASSonline) (https://www.ebi.ac.uk/chembldb/), to predict the cytotoxic activities of NSC777205 and NSC777207 against various cancer cell lines.We analyzed the drug-likeness, PKs, medicinal chemistry, and toxicity of NSC777205 and NSC777207 using SwissADME software (adme.ch) , and com.com/dr) . We usedompounds  to analyompounds  to furthSonline)  created c-MET/EGFR in CRC tissues from The Cancer Genome Atlas (TCGA) database were compared with expression levels in normal tissues using the TNMplot module of the Kaplan-Meier Plotter (https://www.tnmplot.com/). Furthermore, we used the Human Protein Atlas (HPA) database (www.proteinatlas.org) to assess the immunohistochemical (IHC) profile of c-MET/EGFR in tumor samples from CRC patients. We used the MEXPRESS algorithm (https://mexpress.be/index.html) to depict the mechanisms of c-MET/EGFR dysregulation in colorectal cancer (https://precog.stanford.edu/precog_metaZ.datatable.php) , 95% confidence intervals (CIs), and log-rank test p values.Expression levels of l cancer . To analble.php)  to splithttp://www.cbioportal.org/) to mine the Colorectal Adenocarcinoma  dataset for c-MET/EGFR genetic alterations including mutations, copy number alterations, gene mutation co-occurrences, and microbiome signatures and evaluated the prognostic relevance of the alterations in 594 CRC patients using survival analyses of the cohorts (https://www.string-db.org) with the adjusted threshold confidence set to 0.400 (http://timer.cistrome.org/) to analyze correlations of c-MET/EGFR expression with somatic copy number alterations and infiltration of six tumor-infiltrating immune cell subsets  T cells, CD8 T cells, macrophages, neutrophils, and dendritic cells) in CRC. We also used the TIMER server to analyze correlations of c-MET/EGFR expressions and cancer-associated fibroblast (CAF) infiltration. In addition, we analyzed the prognostic relevance of these associations by employing a protocol described in a previous study (http://tide.dfci.harvard.edu)   to analyin vitro anticancer activities against 60 panels of human tumor cancer cell lines representing leukemia (six cell lines), NSCLC (eight cell lines), colon cancer (seven cell lines), CNS cancers (six cell lines), melanomas (nine cell lines), ovarian cancer (seven cell lines), renal cancer (eight cell lines), prostate cancer (two cell lines), and breast cancer (five cell lines) through the National Cancer Institute (NCI). Ranges of 5,000–40,000 viable cancer cells were seeded into each well of 96-well plates and incubated at 37 °C with 5% CO2, 95% air, and 100% relative humidity for 24 h. After incubation, cells were treated with either NSC777205 or NSC777207 at a single dose of 10 μM and further incubated for 48 h : [(Ti−Tz)/(C−Tz)] × 100 for concentrations for which Ti>/ = Tz or [(Ti−Tz)/Tz] x 100 for concentrations for which Ti < Tz.50) (μM) = [(Ti−Tz)/(C−Tz)] × 100 = 50.50% GI  = Ti = Tz.50; μM) = [(Ti−Tz)/Tz] × 100 = −50.50% Lethal concentration  did not meet the required effect on a particular cell line, the value for that parameter was expressed as greater than the maximum concentration tested (>100 μM).In these equations, Tz is the absorbance at time 0; C is the absorbance of the control after 48 h without treatment; Ti is the absorbance of drug-treated cells after 48 h; GIhttps://dtp.cancer.gov/databases_tools/compare.htm) to correlate anticancer activity fingerprints of NSC777205 and NSC777207 with NCI synthetic compounds, standard agents, and molecular targets based on established relationships between cell responses to therapy and gene expression profiles of the cell lines  (http://www.swisstargetprediction.ch/), which operates on the basis of similarity of the queried molecule with known active drugs (http://way2drug.com/dr)  , the Swive drugs , and the.com/dr) .http://avogadro.cc/) (https://pubchem.ncbi.nlm.nih.gov/). All mol2 and sdf files were converted to pdb files using the PyMOL Molecular Graphics System, vers. 1.2r3pre , while pdbqt files of ligands were generated from pdb files using AutoDock Vina  (https://www.rcsb.org/) in protein data bank (PDB) file format and subsequently converted into the Auto Dock Pdbqt format using AutoDock Vina tools. All receptors were charged, hydrogen atoms were added, and water (H20) molecules were removed prior to docking (https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index)  molecular ball-and-stick models in mol2 format of NSC777205 and NSC777207 were obtained using the Avogadro molecular builder and visualization tool vers. 1.XX (dro.cc/) , while 3 States) . Crystal docking . Docking docking . Docking docking  and the p/index) .p value of <0.05. The KEGG and gene ontologies (GOs) were visualized using GraphPad prism software. DTP-COMPARE analyses were conducted using Pearson’s correlations. All survival analyses are presented using Kaplan-Meier plots. GI was calculated relative to cells without drug treatment and the time-zero control. Statistical significance was denoted as *p < 0.05, **p < 0.01, and ***p < 0.001.All statistical analyses were conducted according to each server's instructions. We used the Wilcoxon test to compare statistically significant differences in c-MET/EGFR expressions between CRC cohorts and healthy tissues. Purity adjustment and partial Spearman’s correlations were used to analyze c-MET/EGFR expression correlations with infiltration of CAFs and various immune cells. Gene alteration co-occurrences on the cbioportal were considered significant only at an adjusted p = 1.24e-07) while EGFR expression is lower (p = 4.67e-04) in colorectal cancer cohorts compared to adjacent normal cohorts.  and READ  (p = 0.028) and primary tumors p = 0.04029) but less associated with metastasis or recurrent tumor. Furthermore, both c-MET and EGFR expressions show significant correlation with copy number alterations in colorectal cancer  . Further.01e-14) . We foun.01e-14) . Mean ag.01e-14) . Only 8..01e-14) . Our anal cancer . To assel cancer  and resi+ T cells, B cells, CD8+ T cells, neutrophils, dendritic cells, and macrophages) in CRC tissues  and READ , while MET exhibited no significant correlations with tumor purity in COAD  and READ . Collectively, the above results indicate that c-MET/EGFR expressions were correlated with immune infiltration in COAD and READ  = 0.008, p = 0.015) were risk factors for COAD  and READ . Similarly, c-Met expressions were positively correlated with CAF infiltration in COAD  and READ   . To evalval rate .We mined TCGA PanCancer Atlas dataset for c-MET/EGFR genetic alteration data and evaluated the prognostic relevance of alterations in CRC patients. We found that 3.0 and 4.0% of the 594 CRC patients ) in the dataset respectively harbored genetically altered EGFR and c-MET . Alteratp = 1.82E-05), Actinosynnema (p = 5.63E-05), Castellaniella (p = 8.47E-05), Moorella (p = 9.18E-05), Betaretrovirus (p = 9.45E-05), Pimelobacter (p = 1.25E-04), Arcanobacterium (p = 1.33E-04), and Cryobacterium (p = 1.36E-04) in EGFR non-altered cohorts compared to altered cohorts  enriched genetic alteration co-occurrences with EGFR alterations included SMARCD2, TRIM7, MCMDC2, HNRNPUL1, BEST3, GRB10, PDE6C, and SNX19, while SLC22A2, DST, AKT3, and TAB2 were the only significantly  enriched gene alterations that co-occurred with c-MET alterations  mRNAs in EGFR altered cohorts compared to non-altered cohorts were TRIM7, BARX2, CFAP43, TPBG, HPSE, GNB5, MARK3, FUT8-AS1, SS18, and KCNK1, while SMAD4, MBP, IER3IP1, OXCT1, ALKBH5, C18ORF25, WDR76, TYMS, LINC00909, and GPR3 were overexpressed in c-MET altered CRC cohorts  included cell adherent junctions, EGFR tyrosine kinase inhibitor resistance, central carbon metabolism, CRC, pancreatic cancer, hepatocellular carcinoma, gastric cancer, and melanomas (p < 0.05) included the c-MET, ERBB2, and EGFR signaling pathways, MAPKK activation, and negative regulation of cellular responses to drugs , CD8+ T cell (p < 0.001), neutrophil (p < 0.005), and dendritic cell infiltration in COAD, and macrophage (p < 0.005) infiltration in READ. Arm-level gains of c-MET were positively correlated with CD8+ T cell, B cell, neutrophil, and dendritic cell infiltration in colon cancer and dendritic cells infiltration in READ, while deep deletions of c-MET were associated with CD8+ T cell and neutrophil infiltration of tumors in READ  of a good drug candidate. The compounds were both lead-like and satisfied Lipinski's rules for drug likeness candidates. Furthermore, our in silico analysis suggested that both compounds can be easily absorbed in the gastrointestinal tract and are well permeant to the blood-brain barrier (BBB) especially NSC777205 which demonstrated 2-fold higher permeation of the BBB than NSC777207 based on in silico estimation  and other markers of eco-toxicity also identified both compounds as having low toxicities , brain (Hs683), lung (A549), and bone (SJSA-1) cancer cell lines, with NSC777205 demonstrating higher activities (Pa = 0.31–0.658) than NSC777207 (Pa = 0.303–0.565) against all cell lines (NSC777205 (3-(4-chloro-2-fluorophenyl)-2H-benzo[e]oxazine-2,4(3H)-dione) and NSC777207 (3-(4-chloro-2-fluorophenyl)-7-methoxy-2H-benzo[e]-oxazine-2,4(3H)-dione) were obtained at high purity (>95%) via condensation reactions between salicylic acid and the 2,4-disubstituted aniline moieties of gefitinib to produce a salicylanilide which was further cyclized to the 3-phenyl-2H-benzo[e]-oxazine-2,4(3H)-dione derivatives, NSC777205 and NSC777207, as summarized in timation ; Table 2ll lines .We initially screened NSC777205 and NSC777207 for anticancer activities against the full NCI-60 panel of human tumor cell lines. Interestingly, we found that with single-dose (10 μM) treatment, both NSC777205 and NSC777207 demonstrated antiproliferative activities against all of the NCI-60 cell line panels of breast, prostate, renal, ovarian, colon, melanoma, CNS, leukemia, and non-small cell lung cancers, with NSC777205 demonstrating higher activities than NSC777207. In addition, most of the leukemia (cell death (CD) = 0.77–10.28%), NSCLC (CD = 4.2–50.81%), CNS (CD = 013.71%), melanoma (CD = 0.57–46.24%) renal (CD = 7.01–30.91%), breast (CD = 10.48%), prostate, and ovarian cancer cell lines demonstrated some cytotoxic response to NSC777205 and NSC777207 single-dose treatment, while panels of colon cancer cell lines were completely insensitive to the cytotoxic activity of the compounds at 10 μM. Collectively, these studies demonstrated that both NSC777205 and NSC777207 exhibited a wide spectrum of anticancer activities but demonstrated selective and exclusive antiproliferative activities against all panels of colon cancer cell lines .50 = 11.90–20 µM), both NSC777205 and NSC777207 exhibited high anticancer activities with GI50 concentrations of <6 µM against all cell line panels , A498 (LC50 = 29.60 µM), and UO-31 (LC50 = 51.10 µM) renal cancer cell lines; the SK-MEL-5 (LC50 = 11.30 µM) and UACC-62 (LC50 = 54.10 µM) melanoma cell lines; the SF-295 (LC50 = 57.40 µM) CNS cell line; the COLO 205 (LC50 = 78.40 µM) colon cancer cell line; and the NCI-H522 (LC50 = 95.70 µM) NSCLC cell line  = 47–58) with fingerprints of some NCI synthetic compounds (p = 0.3–0.61) with a number of c-MET/EGFR/PI3K/AKT/mTOR inhibitors, including JNJ38877618, MTX211, SGX-523, altiratinib, TAS-6417, voxtalisib, PI-103 HCL, AMG-458 (c-MET), CC115, and others  docked well into the binding cavities of the receptors with respective binding affinities (ΔG) of −6.90, −6.80 and −6.80 Kcal/mol for EGFR; −8.95, 8.96 and −9.56 Kcal/mol for c-MET; −8.60, −7.90, and −9.0 Kcal/mol for PI3K; and −7.1, −7.6 and −9.2 Kcal/mol for mTOR . The intDespite the availability of various treatment strategies, CRC remains among the top ranked most diagnosed and leading causes of cancer deaths globally . In ordeSMARCD2, TRIM7, MCMDC2, HNRNPUL1, BEST3, GRB10, PDE6C, and SNX19, while alterations of SLC22A2, DST, AKT3, and TAB2 co-occurred with c-MET alterations in CRC. Genetic alterations and concurrent alterations of c-MET/EGFR with other genetic alterations have been well reported in lung cancer  . Tumor-iCAFs are apoptotic-resistant fibroblasts that promote tumorigenic and metastatic properties by secreting cytokines and remodeling the ECM . We founin silico drug-likeness and ADME profiles, and found that both NSC777205 and NSC777207 possessed good drug-likeness properties and obeyed Lipinski’s rule-of-five, a physicochemical-based rule set to evaluate drug candidates for suitability as oral remedy  than NSC777207 (Pa = 0.303–0.565). BBB penetration is an important limiting factor in the use of chemotherapy for treating glioblastomas and other CNS-associated diseases  brain (Hs683), lung (A549) and bone cancer (SJSA-1) cell lines with NSC777205 demonstrating higher activity (Pa = 0.31–0.658) than NSC777207 (Pa = 0.303–0.565). Of importance, among all cell lines, colon cancer cell lines stood out uniquely with all seven cell lines demonstrating strict antiproliferative sensitivity and no cytotoxic response to single-dose (10 μM) treatment of either NSC777205 or NSC777207. This suggests that NSC777205 and NSC777207 would be very useful in growth inhibition and treatment of CRC. Tumors often develop resistance to a wide range of anticancer drugs. Therefore, drug resistance represents an important problem in cancer chemotherapy  was 3-(2-fluoro-4-iodophenyl)-7-methoxy-2H-benzo[e]. In compliance with this recommendation, we used four computational tools including the COMPARE algorithm developed by the USA NCI to identify the most probable targets for NSC7772015 and NSC777207. In the COMPARE analysis, the anticancer fingerprints of NSC777205 and NSC777207 corresponded to anticancer fingerprints of known inhibitors of c-MET/EGFR/mTOR/PI3K pathways. Furthermore, the most highly correlated NCI synthetic compound (-benzo[e] oxazine--benzo[e]. In addi-benzo[e]-oxazin-3-benzo[e]. c-MET p-benzo[e]. It is tin vitro or in vivo validation. Non-covalent interactions such as hydrogen bonds, hydrophobic, ionic, and π-interactions, and van der Waals forces occurring between ligands and receptors are important for stabilization of a ligand within the binding cavity of a receptor (Molecular docking aids the identification of drug lead molecules by unraveling possible interactions between a drug candidate and protein targets, providing structure-activity relationships and therapeutic mechanisms of drug candidates prior to receptor . Our docreceptor . In addireceptor . Both NSBoth NSC777205 and NSC777207 exhibited high similarity with gefitinib and crizotinib, known clinical EGFR and c-MET inhibitors, respectively, in terms of common interacting residues with EGFR and c-MET and similar binding affinities with EGFR, suggesting that both NSC777205 and NSC777207 could be as promising as gefitinib and crizotinib at inhibiting EGFR and c-MET, serving as potential dual targets and thus superseding the strength of individual clinical drugs in arresting cancer proliferation and aggressiveness for which EGFR and c-MET are implicated. This multi-target potency of NSC777205 and NSC777207 could be attributed to the observed activities of NSC777205 and NSC777207 against NCI cell lines that are known to be multi-drug resistant including those that are resistant to more than 10 panels of TKIs .3 group to the salicylic core of NSC777207 was not favorable, as the added moiety did not contribute significant interactions with the receptor-binding residues and also led to overall less-favorable ADMET, physicochemical, and drug-likeness properties and weaker anticancer activities against the panel of NCI cell lines compared to the properties and activities demonstrated by NSC777205 that has no –OCH3 substituent group. However, further in vitro and in vivo analyses in tumor-bearing mice are ongoing in our lab to support this claim and to unravel the full therapeutic efficacies of NSC777205 and 777207 in CRC.Collectively, our study revealed that structural hybridization strategies yielded novel and potential c-MET/EGFR/PI3K/mTOR multi-target compounds, NSC777205 and NSC777205, with promising prospects for treating melanoma, renal, CNS, colon, and NSCLC cell lines. Although NSC777205 and NSC777207 share common interacting residues with the receptors, the overall structure-activity relationship study revealed that the addition of an –OCH3 substituent group from the salicylic core of NSC777205 was responsible for more highly favorable drug-likeness properties and anticancer activities, and more-robust interactions with c-MET/EGFR/PI3K/mTOR than those of NSC777207.In conclusion, our results indicated that, c-Met/EGFR are important biomarker signatures of cancer-associated fibroblasts and tumor immune infiltration, and are of clinical relevance in colorectal cancer. Genetic and epigenetic alterations of c-MET/EGFR were associated with dysfunctional T-cell phenotypes, and poor prognoses of the cohorts. c-MET/EGFR/PI3K/mTOR are therapeutic targets for NSC777205 and NSC777207 with consequent selective cytotoxic preferences for melanoma, renal, CNS, colon, and NSCLC cell lines. However, the removal of an –OCH


Liver cancer cell lines are frequently used in vitro tools to test candidate anti-cancer agents as well as to elucidate mechanisms of liver carcinogenesis. Among such mechanisms is cellular communication mediated by connexin-based gap junctions. The present study investigated changes in connexin expression and gap junction functionality in liver cancer in vitro. For this purpose, seven human liver cancer cell lines, as well as primary human hepatocytes, were subjected to connexin and gap junction analysis at the transcriptional, translational and activity level. Real-time quantitative reverse transcription polymerase chain reaction analysis showed enhanced expression of connexin43 in the majority of liver cancer cell lines at the expense of connexin32 and connexin26. Some of these changes were paralleled at the protein level, as evidenced by immunoblot analysis and in situ immunocytochemistry. Gap junctional intercellular communication, assessed by the scrape loading/dye transfer assay, was generally low in all liver cancer cell lines. Collectively, these results provide a full scenario of modifications in hepatocyte connexin production and gap junction activity in cultured liver cancer cell lines. The findings may be valuable for the selection of neoplastic hepatocytes for future mechanistic investigation and testing of anti-cancer drugs that target connexins and their channels. Liver cancer, mainly represented by hepatocellular carcinoma (HCC), is the second and sixth most common cause of cancer death among men and women, respectively . PrimaryDirect intercellular signaling is mainly mediated by gap junctions . Gap junLiver cancer cell lines are frequently used in vitro tools to test candidate anti-cancer agents as well as to elucidate mechanisms of liver carcinogenesis , such asIn order to assess connexin expression in liver cancer in vitro, a panel of seven cancer cell lines was selected . The panThe cell lines all varied in differentiation status and metastatic potential. In this respect, SK-HEP-1 cells are highly metastatic and display mesenchymal stem cell characteristics , while CMorphology pictures were taken during the exponential growth phase  of the cCx26 mRNA is more restricted to the periportal areas  at a concentration of 0.25 mg/mL. Maintenance medium was replaced 2 h later, after gelatinization of the Matrigel® layer at 37 °C, and again 2 days after addition of the Matrigel® layer. PHH sandwich cultures were maintained for 4 days after seeding.PHH were purchased from KaLy-cell  and cultured in a sandwich culture for scrape loading/dye transfer analysis and immunocytochemistry analysis. PHH were thawed and transferred to a tube with prewarmed cryopreserved hepatocyte recovery medium (CHRM) . After gentle mixing, PHH were pelleted by centrifugation at 100× ® TCP-1011™) was purchased from the American Type Culture Collection  and contained 7 human liver cancer cell lines, namely SK-HEP-1, C3A, SNU-449, SNU-423, SNU-387, SNU-475 and PLC/PRF/5  or Eagle’s Minimum Essential Medium (EMEM)  with addition of 1% MEM Non-Essential Amino Acids Solution  and 1% Sodium Pyruvate  supplemented with 10% fetal bovine serum (FBS)   and 100 units/mL penicillin and 100 µg/mL streptomycin .The Liver Cancer Panel  using a 10× objective. PHH sandwich cultures were imaged daily on the same microscope with a 20× objective. Scale bars were added with ImageJ software (version 1.52p) .TM Mammalian Total RNA Miniprep Kit  combined with an On-Column DNase I Digestion Set  according to the manufacturer’s instructions. RNA yield and purity were assessed using a NanoDrop 2000 spectrophotometer . Next, cDNA was generated with an iScript™ cDNA Synthesis Kit  on a MiniAmp Plus Thermal Cycler  followed by cDNA purification using a GenElute™ PCR Clean-Up Kit . RT-qPCR analysis was performed on an Applied Biosystems QuantStudio 3 Real-Time PCR system . Samples were tested in triplicate and amplified with TaqMan® Gene Expression Assays  targeted towards 5 housekeeping genes, namely actin beta (ACTB), beta-2-microglobulin (B2M), GAPDH, hydroxymethylbilane synthase (HMBS) and ubiquitin C (UBC), as well as towards Cx26, Cx32 and Cx43 . Efficiency was estimated based on a serial dilution of pooled cDNA mix from all samples and 3 no-template controls. Relative alterations compared to PHH were calculated according to the Pfaffl method [+ .The reaction mix contained 2 µL cDNA, 1 µL primer, 7 µL nuclease-free water and 10 µL of TaqManl method  in qbaseg, the resulting pellet was resuspended in the same protein lysis buffer as the cell lines. Next, lysates were transferred to a pre-cooled tube and put on ice for 30 min, during which the lysate was vortexed every 5 min. Supernatants were collected by centrifugation for 20 min at 4 °C and 14,000× g and stored at −80 °C. Protein concentrations were determined with the PierceTM BCA protein assay kit . For immunoblotting, proteins were separated on 10% (Cx43) or 12% (Cx26 and Cx32) Mini-PROTEAN TGX Stain-Free™ precast gels . Samples for detection of Cx26 were heated for 5 min at 95 °C. Gels were blotted onto a nitrocellulose membrane (Cx43 and Cx32)  or a polyvinylidene difluoride membrane (Cx26) . Blocking of the membranes was performed in 5% non-fatty milk in Tris-buffered saline solution (20 mM Tris and 135 mM sodium chloride) with 0.1% Tween-20 for 1 h at room temperature on an orbital shaker. Membranes were incubated overnight at 4 °C with primary antibodies raised against Cx26, Cx32 or Cx43 . Densiometric analysis was performed with Image Lab 6.0.1 software . Signals were normalized against total protein loading and by sum of all data points in a replicate [Membranes were washed 3 times for 10 min and incubated with a secondary antibody  diluted in blocking buffer for 1 h at room temperature. Membranes were washed and visualized by using the Pierce™ ECL Western Blotting Substrate kit  on a ChemiDoceplicate . Data arCell lines and PHH cultures were washed 3 times with ice-cold PBS for 5 min and fixed at −20 °C for 10 min with an acetone–ethanol (1:1) mixture that permeabilized simultaneously. Cells were then washed 3 times for 5 min with ice-cold PBS. Cells were blocked at room temperature with 1% bovine serum albumin (BSA)/5% donkey serum for 45 min and incubated overnight at 4 °C with primary antibodies. Primary antibodies targeted against Cx26, Cx32 and Cx43  were dil2, 5.55 mM glucose and 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); pH 7.4) or CBX buffer (100 µM CBX in SL/DT buffer). Next, a scratch was made with a fine needle in the presence of LY (1 mg/mL) dissolved in SL/DT or CBX buffer. This was incubated for 10 min at room temperature. Then, cells were washed 4 times with Hank’s Balanced Salt Solution (HBSS)/ HEPES buffer  and fixed for 10 min in 4% paraformaldehyde solution. Cells were washed again 4 times with HBSS/HEPES buffer and visualized on a Nikon Eclipse Ti-S microscope  with a 10× or 20× objective. The fluorescent area of the dye spread was measured via ImageJ software (version 1.52p) . Data were normalized to the area of the inhibited cells and expressed as a ratio to the non-inhibited signals in PHH.Cell lines and PHH were washed 3 times with scrape loading/dye transfer (SL/DT) buffer . The number of technical replicates (N) is specified in the figure legends. Statistical analysis was performed in GraphPad Prism v9.0 software , and data are presented as mean ± standard deviation. Normality was assessed by means of a Shapiro–Wilk normality test. Normally distributed RT-qPCR and immunoblot data were analyzed via an ordinary 1-way analysis of variance, followed by a Dunnett’s multiple comparisons test. Scrape loading/dye transfer data were analyzed in the same way when comparing the dye transfer of the cell lines to the dye transfer of the PHH. For comparing the inhibited to the non-inhibited fluorescent area within each cell line, multiple unpaired t-tests were performed with a Holm–Šidák correction for multiple comparisons. Significance levels are indicated by to the following symbols: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001.One batch was used for PHH and all cell lines (