Photobacterium phosphereum T3) and ryegrass (Lolium perenne L.). Compared with Fe-EDTA, the transfer factors of nZVI and B-nZVI were 65.0% and 66.4% lower, respectively. This indicated that although iron nanoparticles accumulated in the roots of ryegrass, they were difficult to be transported to the shoots. The results of this study indicate that B-nZVI has a strong application potential in in situ environmental remediation.The aggregation of nanoscale zero-valent iron (nZVI) particles and their limited transport ability in environmental media hinder their application in environmental remediation. In this study, the Cr(VI) removal efficiency, transport performance, and toxicity of nZVI and bentonite-modified nZVI (B-nZVI) were investigated. Compared with nZVI, B-nZVI improved the removal efficiency of Cr(VI) by 10%, and also significantly increased the transport in quartz sand and soil. Increasing the flow rate can enhance the transport of nZVI and B-nZVI in the quartz sand columns. The transport of the two materials in different soils was negatively correlated with the clay composition. Besides, modification of nZVI by bentonite could reduce toxicity to luminous bacteria ( Nanoscale zerovalent iron (nZVI) is an engineered nanoparticle that is increasingly used in environmental remediation ,2. Due t−1) of nZVI inhibited the rice seedlings growth and inhibited active iron transportation in rice plants [2) to form reactive oxygen species (ROS) [In addition to agglomerate, the potential hazards of nZVI have also raised great concern . As an ee plants . In the e plants ,34,35. Ae plants . Iron ioe plants . Moreovees (ROS) . If excees (ROS) . TherefoThe main aims of this study are: 1) To synthesize nZVI and bentonite modified nZVI (B-nZVI) and investigate the Cr(VI) removal efficiency of these two materials; 2) to investigate the mobility of nZVI and B-nZVI in quartz sand and soil columns; 3) to determine their toxicity toward bacteria and plants. Results of this study could provide a theoretical basis for the application of iron-based nanomaterials in environmental remediation.3∙6H2O), bentonite (Al2O3·4(SiO2)·H2O), anhydrous ethanol, potassium borohydride (KBH4), polyvinylpyrrolidone , and potassium dichromate (K2Cr2O7) were purchased from Aladdin Bio-Chem Technology Co., Ltd., Shanghai, China.Ferric chloride hexahydrate  were dissolved in 100 mL ethanol–water solution (30% V/V) in a three-necked flask. After mechanical stirring under N2 environment for 10 min, 100 mL 0.2 M KBH4 (dissolved in 1% NaOH) was added dropwise by a constant pressure separation funnel. Ferric ions were reduced to zero-valent iron by the following reaction:Nanoscale zero-valent iron (nZVI) and bentonite-supported nZVI (B-nZVI) were prepared through a modified chemical reduction ,39. Brie2 environment for 30 min, the products were washed with anhydrous ethanol for three times. The products were dried under vacuum and sealed storage. The preparation of nZVI used the same procedure, but without bentonite.After mechanical stirring under NThe morphology and elementary composition of nZVI and B-nZVI was characterized by scanning electron microscope  and transmission electron microscopy . Gas absorption operation was carried out for the determination of particles’ specific surface area in a surface area analyzer . X-ray diffraction (XRD) was performed in a X-ray diffractometer  with Cu Kα radiation (λ = 0.154 nm). The scan range was set from 10° to 90° at 40 kV/40 mA. X-ray photoelectron spectroscopy was operated on a X-ray photoelectron spectrometer  with Al Kα radiation (hv = 1486.6 eV) at 12 kV/6 mA.−1 Cr(VI) was prepared by dissolving desired amount of K2Cr2O7, and the initial pH of the solution was adjusted by 0.2 M NaOH or HCl. Cr(VI) removal experiments were performed using conical flasks (500 mL) with an oscillation frequency of 200 rpm. The removal efficiency of Cr(VI)  by nZVI and B-nZVI  was investigated at different initial pH . After 2, 5, 10, 20, 30, 60, 120, 240, 360, 960, and 1440 min of reaction, 2 mL solution was sampled and filtered through a 0.22 μm membrane filter. The concentrations of Cr(VI) in the solutions were determined using the diphenylcarbohydrazide method [A solution with 300 mg L−1, and the background solution was NaCl solution prepared with anaerobic water . The column was first injected with four pore volumes (PVs) background solution in up-flow mode by a peristaltic pump to ensure that the column reaches water saturation and a stable flow state. Subsequently, 4 PV nZVI or B-nZVI suspension and 4 PV background solution were pumped into the column. The suspensions were continuously sonicated to avoid the sedimentation of nZVI and B-nZVI. The effluent was mixed with hydrochloric acid in a ratio of 1:1 with a water bath at 90 °C for 1 h. The total Fe content in the solution was determined by flame atomic absorption .The column used for column transport experiments was a cylindrical plexiglass column with a length of 10 cm and an inner diameter of 1.3 cm. The column transport experiments were carried out in two media: quartz sand (20–30 mesh) and soils . The conPhotobacterium phosphereum T3) was purchased from Institute of Soil Science, Chinese Academy of Sciences . The freeze-dried luminous bacteria were mixed with 1 mL 2% NaCl solution in an ice bath for 2 min. Subsequently, 10 μL of the luminous bacteria solution was added into 2 mL sample solution (prepared with 3% NaCl). After being incubated at 20 °C for 15 min, the bioluminescence intensity was measured by DXY-2 microtox test instrument . Each sample was determined at least three times. HgCl2 was used as a reference in this test, and the relative fluorescence intensity had a linear relationship with the concentration of HgCl2  [−1) was evaluated in addition to the nZVI and B-nZVI treatments.The luminous bacteria (ctively) . The toxLolium perenne L.) seeds were surface sterilized in 3% hydrogen peroxide solution for 30 min and thoroughly washed with deionized water. Then the ryegrass seeds were transferred to moist gauze and incubated in darkness at 28 °C for 6 days. The 6-day-old seedlings were transferred to a hydroponic solution with 1/3 strength heavy elements (1 mM Ca(NO3)2, 0.5 mM Ca(H2PO4)2, 0.5 mM K2SO4, 1 mM MgSO4, and 1.5 mM NH4NO3) and full strength trace elements . The hydroponic solution was adjusted to pH 5.8. Different doses of nZVI and B-nZVI  were added into the hydroponic solution for the treatment groups. Control  and ionic treatments using Fe-EDTA  were prepared in addition to the nZVI and B-nZVI treatments; three parallels for each treatment, and 24 seedlings for each parallel. The plant culture was performed in a growth chamber at 25 °C for 16 h in the day and at 20 °C for 8 h in the night with 50–60% relative humidity. The hydroponic solution was replaced every 3 days.Selected uniform perennial ryegrass . Fresh plant samples were mixed with 4 °C phosphate buffer (PBS), homogenized in a glass homogenizer. The homogenate was centrifuged at 4 °C, and the supernatant was separated for the determination. SOD activity assay kit  and catalase assay kit  were used for enzyme activity assay. Lipid peroxidation in plants was determined by MDA content by 2-thiobarbituric acid (TBA) reaction .After 21 days of growth, ryegrass was harvested and washed with deionized water. Then shoots and roots were separated, and their dry weights were measured. The contents of Fe in plants were determined by ICP-MS  after being digested with HNO2/g) to 26.5 m2/g, and the adsorption/desorption average pore diameters of nZVI and B-nZVI were 16.6/6.1 nm and 16.2/8.4 nm, respectively. As observed from the XRD patterns of nZVI and B-nZVI  facet of bcc iron (JCPDS no. 06-0696) [θ of 19.8°, 35.8°, and 62.8° can correspond to montmorillonite (JCPDS no. 29-1498) [θ of 27.8° was observed in the pattern of B-nZVI, which is consistent with the characteristic peak of silicon oxide (JCPDS no. 82-1566), one of the main components of bentonite.The morphology and size of the newly prepared nZVI and B-nZVI were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) . The SEMd B-nZVI , a diffr06-0696) . No othe0 and Cr(VI) is a process of consuming hydrogen ions, and thus the increased concentration of hydrogen ions in the solution can promote the reaction to proceed to the positive direction [In order to investigate the Cr(VI) removal efficiency of nZVI and B-nZVI, the removal experiments of Cr(VI) were studied under different initial pH conditions . The results showed that for both nZVI and B-nZVI, the removal process of Cr(VI) was very fast . The remirection . In addiirection ,47.3/2 and Cr 2p1/2 photoelectron peaks at 576.8 eV and 586.5 eV represent that Cr(OH)3, Cr(OH)O, and Cr2O3 are the main species of Cr [2Cr2O7 [3/2 and Fe2p1/2 are 710.9 eV and 724.5 eV, respectively. This indicates that Fe in the product basically exists in the form of Fe(III), and the possible chemical structure is iron oxide hydrate (FeOH), magnetite (Fe3O4), and hematite (Fe2O3) [0 after the reaction, and no such photoelectron peak was found in the spectra of B-nZVI. Li et al. also found that Fe0 can be still detected in the nZVI after reacting with Cr(VI) for 24 h [0 more sufficient, thus Fe0 was completely oxidized to Fe(III) after the reaction.Further analysis of the reaction products by XPS showed that the reaction products were mainly composed of Fe, Cr, O, and C elements . The Cr es of Cr . The spies of Cr ,49. In a [2Cr2O7 . This ma (Fe2O3) . In addifor 24 h . This ca−1 to 3.0 cm min−1, the plateaus increase from 0.02 to 0.04. Previous studies have also found that increasing the flow rate can improve the mobility of nanoparticles, mainly because increasing the flow rate can generate stronger hydrodynamic forces to keep the colloids in the mobile phase [The transport behaviors of nZVI and B-nZVI were studied by column transport experiments. The breakthrough curves of two materials begin at 1 PV and reach their plateaus at about 1.5 PV . For nZVle phase ,55.−1, the breakthrough plateaus can reach 0.35, which is about eight times that of nZVI. The attachment of nZVI to bentonite decreased the magnetic attraction and created steric repulsion between nanoparticles, thus facilitating the transport of nZVI through quartz sand column [After modification with bentonite, the mobility of nZVI in quartz sand column has been greatly improved b. At thed column . Moreoved column . Similard column .The mobility of nZVI and B-nZVI in soil columns packing with three different soils was studied . Similar−1 of nZVI can reduce the relative bioluminescence intensity by 93.7%, which indicates that high concentration of nZVI is highly toxic to the luminous bacteria. Some studies reported that nZVI had no cytotoxicity toward microorganisms. For example, 1000–10,000 mg/L of nZVI had no effect on the survival and activity of Klebsiella planticola, and did not cause significant cell damage [Escherichia coli were inactivated with the treatment of 70 mg/L nZVI [Escherichia coli, Bacillus subtilis showed a stronger tolerance to nZVI, but the survival rate of Bacillus subtilis also decreased with the extension of nZVI treatment time [−1 treatments. However, the relative bioluminescence increased from 6.3% to 14.6% with the treatment of 500 mg L−1, indicating that the toxicity of B-nZVI was lower than nZVI. In the study reported by Qiu et al. [−1 nZVI can decrease the relative bioluminescence intensity of luminous bacteria by 49.3%, and the generated iron ions were considered to be the main source of toxicity. In order to figure out the effect of iron ions to luminous bacteria, Fe-EDTA treatments were prepared as ionic controls. Compared with nZVI and B-nZVI, the treatment of 50 mg L−1 Fe-EDTA had no significant effect on the relative bioluminescence intensity. The toxicity of Fe-EDTA to luminous bacteria was produced as the treated concentration was increased. However, compared with the same concentration of nanomaterials, its toxicity was significantly reduced. This suggests that the toxicity of nZVI and B-nZVI to luminous bacteria may not only come from the dissolved iron ions, but also from the nanoparticles themselves. Metal-based NPs have been found to induce perturbations on microbes not only by the dissolved ions, but also by the particles themselves [2−, and H2O2 [The toxicity of nZVI and B-nZVI was evaluated by luminous bacteria toxicity test . The rell damage . Howeverg/L nZVI . Compareent time . Surfaceent time . After tu et al. , the preemselves . Due to and H2O2 . The excand H2O2 .−1 of nanomaterials had no significant effects on the biomass of the plants  after 21 days of hydroponic culture. Although iron is one of the essential nutrients for plants, excessive iron concentration can inhibit plant growth [The phytotoxicity of nZVI and B-nZVI was investigated by hydroponic experiment of ryegrass. The results indicated that the addition of 50 and 200 mg Le plants . Howevert growth .−1 nanomaterials was 10.5 times and 5.6 times that of the control group, respectively. The Fe accumulation in the shoots also increased by 2–3 times compared with the control group, while the Fe content in the group treated with B-nZVI was relatively independent of the dosage of B-nZVI. Similarly, the total Fe content in the plants with the treatment of Fe-EDTA was higher than in the control group. However, compared with the same concentration of nanomaterials treatment groups, the Fe-EDTA treatment group had a lower Fe content in the roots. As a result, the translocation factor (Cshoot/Croot) of the Fe-EDTA treatment group was higher than that of the nanomaterials-treated groups  of nZVI has also been found to up-regulate the CAT activity and MDA content in rice plants [Overall, the treatments of iron-based NPs had greater effects on the roots of plants rather than the shoots. The results of antioxidative enzymes and lipid peroxidation correlated well with the results of biomass and Fe contents. Excessive iron accumulation can inhibit plants growth, cause oxidative stress, and stimulate plants to up-regulate antioxidative enzymes activity. Oxidative stress caused by iron-based NPs has been reported in other research. The exposure of 100 mg Ln plants . High doe plants .Photobacterium phosphereum T3) and perennial ryegrass (Lolium perenne L.). The transfer factors in nZVI and B-nZVI treatments were much lower than those in the Fe-EDTA treatments. This indicated that although iron nanoparticles accumulated in the roots of ryegrass, they were difficult to be transported to the shoots. The results show that bentonite is an ideal carrier, which can not only enhance the removal ability of nZVI to Cr(VI) and improve its migration performance in porous media, but also reduce its ecological toxicity. B-nZVI has a strong potential in the remediation of groundwater and soil polluted by Cr(VI).In this study, it was found that modification of nZVI by bentonite could improve the removal efficiency of Cr(VI) by 10%. The transport of nZVI was limited both in quartz sand and soil columns, and the transport performance can be improved by increasing the flow rate. Compared with nZVI, the transport of B-nZVI in quartz sand and different types of soil was increased by 8 times and 2–10 times, respectively. The transport of the two materials in different soils was positively correlated with the sand composition. Modification of nZVI by bentonite could reduce the toxicity to luminous bacteria (


Bothrops jararaca snake venom, as well as the functional implications of the proteomic changes. Cell lines were treated with sub-toxic doses at either 0.63 μg/mL (low) or 2.5 μg/mL (high) of B. jararaca venom for 24 h, conditions that cause no cell death per se. Proteomics analysis was conducted on a nano-scale liquid chromatography coupled on-line with mass spectrometry (nLC-MS/MS). More than 1000 proteins were identified and evaluated from each cell line treated with either the low or high dose of the snake venom. Protein profiling upon venom treatment showed differential expression of several proteins related to cancer cell metabolism, immune response, and inflammation. Among the identified proteins we highlight histone H3, SNX3, HEL-S-156an, MTCH2, RPS, MCC2, IGF2BP1, and GSTM3. These data suggest that sub-toxic doses of B. jararaca venom have potential to modulate cancer-development related protein targets in cancer cells. This work illustrates a novel biochemical strategy to identify therapeutic targets against cancer cell growth and survival.Cancer is characterized by the development of abnormal cells that divide in an uncontrolled way and may spread into other tissues where they may infiltrate and destroy normal body tissue. Several previous reports have described biochemical anti-tumorigenic properties of crude snake venom or its components, including their capability of inhibiting cell proliferation and promoting cell death. However, to the best of our knowledge, there is no work describing cancer cell proteomic changes following treatment with snake venoms. In this work we describe the quantitative changes in proteomics of MCF7 and MDA-MB-231 breast tumor cell lines following treatment with  Bothrops genus [Bothrops jararaca (B. jararaca) snake is a complex mixture composed of proteins, peptides, amino acids, nucleotides, lipids, and carbohydrates that present a range of different actions when they are isolated or together [Bothrops venoms [Bothrops venoms are composed of various classes of toxin, including metalloproteinases, serine proteinases, phospholipases A2, and C-type lectins, the most abundant components participating in the local and systemic envenomation effects.Ophidian accidents constitute a serious public health problem in Brazil, with an average of 29,000 cases and 125 deaths reported every year  [together ,4,5,6,7,together ,9. Severs venoms ,14,15,16B. jararaca engenders three main activities: proteolytic, coagulant, and hemolytic. The proteolytic activity causes degradation of extracellular matrix proteins, plasma, and cell surface [B. jararaca venom is composed of a complex mixture of proteins, mainly metalloproteinases, serine proteinases, phospholipases (PLA2), and L-amino acid oxidases. The metalloproteinases comprise most of the venom composition [The venom of  surface , which r surface ,18. In a surface . About 9position ,21. Theyposition ,23. In tposition . The thiposition . Finallyposition , are flaposition ,28. Howeposition .Echis carinatus snake venom and produced by Millennium Pharmaceuticals and Schering-Plow. It is used as an antiplatelet drug [B. jararaca venom. It is used for the treatment against hypertension and renal insufficiency [2+, inducing the release of cytochrome C, decreasing or increasing the expression of proteins that control the cell cycle, and causing damage to cell membranes [Snake venom constituents have been isolated and studied for their therapeutic potential in the treatment of various diseases. One example is Eptifibatide, marketed as Integrilin, derived from the let drug . Anotherficiency ,32. In aficiency , severalembranes ,34,35,36embranes .With the rapid advances of nano-scale liquid chromatography (nLC) and mass spectrometry (MS) technologies in the last two decades, nLC-MS/MS-based proteomics analysis has been widely applied as a powerful tool for biomarker discovery to improve cancer therapy ,39. A nuB. jararaca snake venom treatment on breast cancer-related cell proteome. In the present study, nLC-MS/MS was used to characterize the effects of sub-toxic doses of B. jararaca snake venom on two different breast cancer cell lines MCF7 and MDA-MB-231. MCF7 and MDA-MB-231 are non-metastatic and metastatic tumor cell lines, respectively. They are characterized by a high degree of glycolytic efficiency that promotes the interaction between the tumor cell and the extracellular matrix [B. jararaca snake venom in these cell lines highlight proteins and cell pathways that could be targeted in cancer therapy.However, to the best of our knowledge, there has been no report that describes the effects of r matrix . Althougr matrix ,48. ProtB. jararaca venom cytotoxicity assay on MCF7 and MDA-MB-231 cell lines was monitored using the WST-1 reagent. This analysis showed that although cell viability was similar between both MCF7 and MDA-MB-231 cell lines, they had different venom resistance profiles where MDA-MB-231 cells showed to be more resistant to the venom when compared to the MCF7 cells. Although both cell lines started to die at doses higher than 2.5 μg/mL, at the 5.0 μg/mL of venom, only about half of the MDA-MB-231 cells have died while most of the MCF7 have died at this venom concentration , 725 proteins were identified in cells treated with 0.63 µg/mL of venom, and 713 proteins were identified in the cells treated with 2.5 µg/mL of venom. Comparative analysis of the identified proteins showed that among all of the 789 proteins identified, 637 proteins were in common to all conditions, 657 proteins were in common between the control and the 0.63 µg/mL venom treatment, 656 proteins were in common between control and the 2.5 µg/mL venom treatment, and 677 proteins were in common between the 0.63 µg/mL and 2.5 µg/mL venom treatments  of proteins whose abundance increased more than 1.5× in the MCF7 cell line treated with 2.5 μg/mL of venom showed a high interconnection of proteins related to metabolic process (in red) and metabolism pathways (in blue) a. InterePPI analysis of the MCF7 cell lines with FC ≥ 1.5 treated with 0.63 μg/mL and FC ≤ 0.67 when cells were treated with 0.63 μg/mL and 2.5 μg/mL of venom are shown in We further analyzed proteins exclusively identified in one or two of the three conditions in both MCF7 cell line  and MDA-Of the proteins identified exclusively when cells were treated with 0.63 μg/mL of venom there were 28 proteins from MCF7 cell line, from which we highlight Cytochrome b-c1 complex subunit 1, mitochondrial (UQCRC1), Latexin (LXN), Vesicle-associated membrane protein-associated protein B/C (VAPB), Cysteine and glycine-rich protein 1 (CSRP1), and DDB1- and CUL4-associated factor 7 (DCAF7). From MDA-MB-231 cell line, 20 proteins were identified from which we highlight Eukaryotic translation initiation factor 2 subunit 2 (EIF2S2), low molecular weight phosphotyrosine protein phosphatase (ACP1), Transcription factor A, mitochondrial (TFAM), and Rae1 protein homolog (RAE1) .Proteins identified exclusively at the high dose treatment of 2.5 μg/mL of venom included 17 proteins detected in the MCF7 cell line, from which we highlight 26S proteasome non-ATPase regulatory subunit 6 (PSMD6) and 60S ribosomal protein L36 (RPL36). Similarly, we identified 44 proteins in MDA-MB-231 cell line from which we highlight protein Ubiquitin carboxyl-terminal hydrolase (HEL-117) .B. jararaca venom. From the MCF7 cell line, we identified 40 proteins from which we highlight: Serine/threonine-protein kinase ATR (ATR) and Jupiter microtubule-associated homolog 1 (JPT1). From the MDA-MB-231 cell line, we identified 22 proteins from which we highlight Angiomotin (AMOT) and Small nuclear ribonucleoprotein G (SNRPG)  . Among the proteins that were identified in the control group and at the 2.5 μg/mL venom treatment, we highlight: Small nuclear ribonucleoprotein Sm D1 (SNRPD1) in MCF7, and Epididymis secretory protein Li 71 (Hel-S-71) and Isoform Far upstream element-binding protein 3 (FUBP3) in MDA-MB-231 cell line . Of protB. jararaca venom treatment of MCF7 and MDA-MB-231 cells, including SNX3, HEL-S-156an, UQCRC1, RPL36, and ATR identified in MCF7 cell line and H3C15/HIST2H3, HEL-S-132P, MTCH2, TFAM, KCTD12, RPL34, and RPL37A identified in MDA-MB-231 cell line, and histone H3F3B and LAP3 in both cell lines.The present study successfully identified cancer-related proteins that undergo significant changes upon B. jararaca snake venom [With the discovery of rattlesnake venom’s antitumor activity in 1931 by Essex and Priestley  and lateke venom ,117,118,ke venom ,120.2+ influx. Several studies have revealed that these enzymes are critical regulators of cancer pathologies in several types of cancers including breast cancer [B. jararaca venom may have an important antitumor effect on Ehrilich ascites tumor cells in vivo and in vitro [Snake venoms have a complex mixture of proteins that can account for 95% of the total dried weight. Metalloproteases, serine proteases, LAAOs, and PLA2s are some of the most abundant proteins present in bothropic venoms, and the toxic action of these proteins are responsible for myotoxic effects, disruption in the coagulation cascade through thrombin-like proteins (serine proteases), imbalance in the blood homeostasis by the action of some metalloproteinases, apoptosis induction, and changes in the protein expression in oxidative stress and energy metabolism by the increase of Cat cancer ,126,127.in vitro .Aiming at the drug development, several groups have used proteomics approach to observe up- or down-regulated effects of proteins resulting from disease activity or side effects of the treatments ,130,131.Calloselasma rhodostoma, Macrovipera lebetina and Bothrops mattogrossensis significantly inhibit cell viability, either with crude or fractionated venoms and in different tumor cell lines such as LS174T , HCT116 , HT29 , HEL92.1.7 (erythroleukemia), and SK-BR-3 (breast adenocarcinoma) cell lines [Proteomics studies using tumor cell lines has been performed to systematically characterize protein complexes that may be important in disease or drug development, comparing the protein levels of cells in normal versus pathological situations . Studiesll lines ,134.B. jararaca venom, we tested two different invasive breast cancer cell lines with different phenotypic and genotypic differences, MCF7 and MDA-MB-231 cell lines. They were treated with different venom concentrations ranging from 0.32 µg/mL to 20 µg/mL. When treated with over 10 µg/mL of venom both MCF7 and MDA-MB-231 cell lines showed rounded and irregular morphologies and the MCF7 cell line detached from the plate. These results are similar to the results observed by Bernardes-Oliveira et al. (2016) when testing 50 µg/mL of venom from B. jararaca and Bothrops erythromelas for 48 h on SiHa HPV-16 and HeLa cells, both derived from cervical tumors. They observed that the cells became more rounded, with gradual size reduction and detachment from the cell monolayer [B. jararaca venom treatment. Based on both morphological and cell viability analyses, we selected the concentrations of 0.63 µg/mL (low sub-toxic dose) and 2.5 µg/mL (high sub-toxic dose) for further proteomic analyses.To evaluate the cytotoxicity of onolayer . In addiB. jararaca venom presented less proteins with FC ≥ 1.5 when compared to the MCF7 cell line. We identified 137 proteins with FC ≥ 1.5 at 2.5 μg/mL venom treatment in MCF7 while 34 proteins in MDA-MB-231 at 2.5 μg/mL venom treatment. At the low 0.63 μg/mL venom treatment, 25 proteins in MCF7 presented FC ≥ 1.5 and 16 proteins in MDA-MB-231. However, MDA-MB-231 showed more proteins with FC ≤ 0.67 compared to MCF7 cell lines. MDA-MB-231 showed 28 proteins presenting FC ≤ 0.67 at 0.63 μg/mL venom treatment and 19 proteins in MCF7; and 41 proteins with FC ≤ 0.5 at 2.5 μg/mL venom treatment in MDA-MB-231 cell lines compared to 23 proteins in MCF7 cell line. Among these proteins, our data showed that B. jararaca venom was able to modulate, up or down, the abundance of several proteins playing essential roles in tumorigenicity. For example, the venom treatment increased the abundance of SNX3, HEL-S-156, MCCC2, and GSTM3 in the MCF7 cell line, and H3C15/HIST2H3 and MTCH2 in the MDA-MB-231 cell line whose overexpression has been related to increase in tumorigenicity  increased more than 1.5×, and proteins that abundance decreased less than 0.67×, after 24 h treatment with 2 in RPMI 1640 culture medium  supplemented with 10% inactivated fetal bovine serum  and 25 mg/mL ampicillin and 100 mg/mL streptomycin antibiotics. Cells were split when the confluence reached 80%.MCF7 and MDA-MB-231 breast cancer cell lines were previously acquired from ATCC  and maintained at our laboratory cell bank. Cells were thawed and cultured at 37 °C in 5% COB. jararaca (lot 01/09-2) used in this study was a pool made from the extraction of 697 snakes collected at various Brazilian locations . Extracted venom were pooled and lyophilized at the department of herpetology at Butantan Institute under the coordination of Dr. Marisa Maria Teixeira da Rocha, and assigned for use and testing at the Laboratory of Applied Toxinology at Butantan Institute. The methods and use of venom in this work were approved by Butantan Institute Ethics Committee under the certification CEUAIB #9766150719 (2019).The venom of 4 cells were plated in each well of a flat bottom 96-well microtiter plate. Cells were cultured at 37 °C in 5% CO2 and on the day prior to analysis (70–80% confluency), culture medium from each well was exchanged to 100 μL of medium in the absence or presence of venom in different concentrations. After 24 h, 10 μL of the WST-1 reagent previously dissolved into an Electro Coupling Solution (ECS) were added to each well and incubated for 4 h at 37 °C in 5% CO2. The absorbance at 450 nm of each well was measured using a microplate reader FlexStation 3 spectrophotometer . The determination of the absorbance and the quantification of viable cells were calculated using SoftMax Pro  and Microsoft Excel software.The cytotoxicity assays were carried out by treating the cells with different concentrations of venom in the range from 0.1 μM to 20 μM. The cytotoxicity tests were performed using the colorimetric method of cell viability analysis using the WST-1 cell proliferation reagent kit  according to manufacturer’s instructions. Briefly, 105 cells were plated and after the cells had reached a confluence of 70–80% (2–3 days), different concentrations of B. jararaca venom were added to the culture medium, starting from the cell cytotoxicity threshold up to a 1000-fold dilution for 24 h. For mass spectrometry analysis, cells were washed with ice-cold PBS and lysed with 1 mL ice-cold 8 M urea supplemented with cOmpleteTM protease and phosphatase inhibitors cocktail . Fifty microliters of the cell lysate were separated for protein quantification using BCA  and the lysate were stocked at −80 °C until further preparation of the sample for mass spectrometry analysis. Both cytotoxicity and proteomics experiments were performed in three biological replicates.In a six-well plate, 2 × 10To the cell lysates, four microliters of 10 mM dithiothreitol (DTT) were added to reduce the disulfide bonds and incubated at 56 °C for 1 h. After incubation with DTT, 40 μL of iodoacetamide (IAA) was added for the alkylation of the cysteines for 1 h in the dark at room temperature. After incubation, 2.5 mL of ammonium acetate and 20 μL of trypsin  were added enough to have an enzyme:protein ratio of 1:50 and incubated at 37 °C overnight. The reaction was stopped by adding 30 μL of 100% acetic acid. Samples volume were reduced in a SpeedVac  until the volume has lowered to 50 μL and were further desalted on in-house manufactured stop-and-go extraction tips (Stage-Tip) with three SDB-XC  membranes as previously described . The stam/z, and a dynamic exclusion of 70 s. Protein identification was performed using Mascot , MaxQuant , and Peaks Studio  against the Homo sapiens database downloaded from the Uniprot in March 2019 (www.uniprot.org accessed on 1 October 2019). As search parameters, oxidation of methionine was set as a variable modification and carbamidomethylation of cysteine was set as a fixed modification. Searches were performed with mass error tolerance of 10 ppm for MS and 0.3 Da for MS/MS, and trypsin was selected as the proteolytic enzyme used in the digestion of proteins, and up to two miscleavages were allowed. Mass spectrometry-based proteomic analysis of MCF7 and MDA-MB-231 cell lysates, treated or not treated with low or high doses of B. jararaca venom were performed in triplicate. All raw data files for these analyses were uploaded and are available at: http://massive.ucsd.edu/MSV000084138/ accessed on 5 July 2021.Dried samples were resuspended in 0.1% formic acid and analyzed on LTQ-Orbitrap Velos mass spectrometer  coupled to an EASY II nano liquid chromatographer (Thermo Scientific) using the shotgun approach. The spectrometer was equipped with a nanospray source connected to an in-house prepared analytical column (10 cm × ID 75 μm × OD 360 μm) packaged with 7 cm of 5 μm C18 resin . Precolumn (7 cm × ID 75 μm × OD 360 μm) was also prepared in-house packed with 5 cm of 10 μm C18 resin . The LC-MS/MS analyses were carried out by injecting 5 μg of the peptide extract and the peptides eluted from the column with a gradient of 5–40% in 100 min of solvent B  at a flow rate of 200 nL/min. The nanospray source was operated at 1.8 kV. The peptide mixture was analyzed by the acquisition of spectra in the full scan mode at a resolution of 30,000 for the determination of molecular masses (MS) of up to 10,000 Da. The 10 most intense peaks were automatically selected via data dependent acquisition (DDA) for the subsequent acquisition of spectra of the ions product to MS/MS for amino acid sequence determination at a resolution of 7500, a maximum injection time of 30 ms, a range of 200 to 2000 geneontology.org accessed on 1 October 2019) and PantherDB (pantherdb.org accessed on 1 October 2019). The proteins presenting different profiles in the venom conditions were also analyzed using KEGG (www.genome.jp/kegg accessed on 1 October 2019) canonical pathways to determine the protein group or pathways that have undergone changes with different concentrations of the venom treatment. The PANTHER Classification System  is a research tool that covers evolutionary and functional information on protein family genes and has been widely used to understand protein evolution and its functional classification [B. jararaca venom that presented FC ≥ 1.5 in order to identify an enrichment pattern of protein families and pathways affected by the venom in the cell lines.The entire proteome was analyzed to classify protein profiles by biological processes, molecular function, cellular components, and cellular pathways using Gene Ontology . The set of protein dissimilarities were computed using the “Euclidean” distance with the function “dist” to the hierarchical clustering based on the package and function “hclust”. There was employed the agglomerative method with “ward.D2”. The fold change and the protein–protein interactions provided by information from the String database [B. jararaca venom using the Homo sapiens reference genome.Semi-quantitative protein analysis was performed using the Label Free Quantification MaxLFQ algorithm from MaxQuant software with an FDR rate of ≤1% to compare relative abundance of proteins in each of the cell lines . Data gedatabase  was usedhttp://www.sthda.com/english/rpkgs/factoextra accessed on 1 November 2020) for graphical visualization.Principal component analysis (PCA) was applied to find which linear combinations of the differentially expressed proteins with ≥1.5 would explain most of the variability for the different cell lineage conditions. The PCA analysis was performed using the R-statistics packages FactoMineR (accessed on 1 November 2020)  and Fact


The differences between the two cell lines are a good model for the remarkable heterogeneity of human squamous cell carcinoma. Study of the metastatic potential of these models revealed that both cell lines represent the organotropism of LUSC in humans, i.e. affinity to the brain, bones, liver and adrenal glands. In summary, we have generated valuable cell line tools for LUSC research, which recapitulates the complexity of the human disease.There is a paucity of adequate mouse models and cell lines available to study lung squamous cell carcinoma (LUSC). We have generated and characterized two models of phenotypically different transplantable LUSC cell lines, i.e. UN-SCC679 and UN-SCC680, derived from A/J mice that had been chemically induced with N-nitroso-tris-chloroethylurea (NTCU). Furthermore, we genetically characterized and compared both LUSC cell lines by performing whole-exome and RNA sequencing. These experiments revealed similar genetic and transcriptomic patterns that may correspond to the classic LUSC human subtype. In addition, we compared the immune landscape generated by both tumor cells lines  Summary: Generation and characterization of two phenotypically different and transplantable mouse model lung squamous cell carcinoma cell lines – UN-SCC679 and UN-SCC680 – to be used in different research scenarios, including immunotherapy. Lung cancer is the leading cause of cancer-related death worldwide . The incOf all lung cancers ∼20-30% are classified as lung squamous cell carcinoma . There are two types of lung cancer, i.e. small lung cell cancer (SCLC) and non-small cell lung cancer (NSCLC), and LUSC is more strongly associated with tobacco smoking than any other subtype of NSCLC. Age, exposure to second-hand smoke, asbestos, mineral and metal dust, and radon constitute other risk factors for LUSC.Basal stratified squamous lung epithelial cells are the most likely origin for LUSC . Within LUSC is a less-studied NSCLC subtype than LUAD. Different factors have contributed this fact. First LUSC biology is more challenging than that of LUAD and, on average, human LUSCs have more mutations per megabase than LUAD . Thus, LIn this present work, we performed exhaustive molecular and functional characterization, and comparison of two novel LUSC research cell line models. These models consist of two A/J mouse-derived syngeneic cell lines obtained by using NTCU-induced LUSCs. These cell lines might become robust tools for the study of squamous cell lung cancer in a reliable and reproducible manner, and when testing novel antimetastatic and immunotherapy agents. In fact, one of the two cell lines (UN-SCC680) has successfully been used by our group to evaluate a strategy of combined immunotherapy . Here, win situ carcinomas were observed in mouse bronchi upon NTCU treatment . Progressive de-differentiation of the typical squamous phenotype was observed with increasing passages, as previously reported for human lung LUSC cancer cell lines . We compared the differentially expressed genes identified in UN-SCC679 with those in UN-SCC680 cells. We observed that ∼80% of the differentially expressed genes are present in both cell lines. This indicated that, despite their differences in mutational patterns, UN-SCC679 and UN-SCC680 cells share many molecular similarities that reflect their identical squamous origin. Shared and non-shared differentially expressed genes are listed in Table S5. Compared to normal lung tissue, we found that both cell lines are enriched in p63  and that expression of genes related to the classic subtype described for human LUSC genomic subtypes, such as Gsto1, Aldh3a1, Bcl6, Atp5g3, Dld, Odc1, Trp63, Gsta4, Ndufb5, Ephx1 or Cox5b (data not shown) is increased . Finally, both cell lines overexpress Arnt2, which has been previously associated to lung cancer , UN-SCC679 cells showed an advantageous in vivo tumor growth in immune-competent mice compared to that of UN-SCC680 cells , i.e. anti-PD-1 therapy . Taking 80 cells A,B, sugg80 cells A. These 80 cells B, the reThe success of checkpoint blockade in NSCLC, both squamous cell carcinoma and adenocarcinoma, is strongly influenced by the nature of the immune infiltrate present in the tumor . To bettWe completed our immune population study by performing a multiplexed immunohistochemistry experiment using VECTRA technology D and E. 5 UN-SCC679 or UN-SCC680 cells that had been transfected with a triple modality GFP-luciferase-thymidine reporter gene . To explore the specific metastatic potential of the cell line models, we inoculated the left heart ventricle of A/J mice with 1×10ter gene A. Thus, ter gene B. At the has been shown to promote proliferation and cell autophagy via ERK1/2 signaling in human lung cancer cells (DNAH6) belongs to the dynein family, whose members encode large proteins that constitute the microtubule-associated motor protein complex; it has recently been related to smoking-associated lung cancer (CDH23) – a member of the cadherin superfamily, whose genes encode calcium-dependent cell-cell adhesion glycoproteins – is downregulated in lung cancer, working as a suppressor of cell migration (CSMD3) to be a frequently mutated gene in human LUSC, resulting in an increased proliferation of airway epithelial cells (FBN1), encoding an extracellular matrix glycoprotein that serves as a structural component of calcium-binding microfibrils, seems to play an important role in tumor-related immune infiltration and has been proposed as a prognostic and predictive biomarker for immune therapy against pancreatic ductal adenocarcinoma (PDAC)  . Finallya (PDAC) . Our stuRelevant LUSC studies coincide in emphasizing the molecular heterogeneity of this tumor type, which makes the development of targeted therapies very difficult . Thus, wThe National Comprehensive Cancer Network guidelines for LUSC treatment  do not rKRAS and P53 are partly responsible for the resistance of lung tumors to immunotherapy , which could be involved in its resistance to anti-PD-1 treatment. However, UN-SCC680 cells, which responded to immunotherapy to some extent, contained mutated Kras. Nevertheless, the specific Kras mutation found in UN-SCC680 cells was a rare alteration of the gene, which differed to the classic G12V/D mutation that characterizes lung cancer. This detail must be taken into account, since Kras is not a gene characteristically mutated in LUSC and because the mutation we found in our cell line might have other functional and biological implications regarding the tumoral context, which are different to those expected with the classic G12V/D mutation.It is well stablished how specific mutations drive immune evasion in cancer. Specifically, mutations in otherapy . UN-SCC6In the context of sensitivity to immunotherapy and based on the tumor immune landscape, six immune subtypes (ISs) have been identified in SCC. The most common for LUSC – comprising ∼41% of cases – is IS1, followed by IS5 (25%) and IS3 (14%). Both IS1 and IS3 are ‘immune-cold’, characterized by a low lymphocyte infiltration rate, a high immunosuppressive microenvironment and a poor prognosis. IS3 has the worst outcome among all subtypes . AccordiThe immune landscape and mutational burden have been shown to predict the response of LUSC to immunotherapy. In general, a tumor with a high lymphocytic infiltrate and an elevated mutational burden will have a greater response to immunotherapy treatments . UN-SCC6A well-known feature of human lung tumors is their selective metastatic tropism. Lung cancer most commonly spreads through bloodstream to the liver (34.3%) and adrenal glands (32.6%), followed by bones (14.9%) and other organs, such as the central nervous system (CNS) (12%), kidney (10.9%), myocardium (9.1%), pancreas (5.1%), spleen (4%), small or large intestines (3.4%), stomach (2.3%), thyroid gland (1.7%) and ovary (0.6%) . The twoIn conclusion, we have generated and thoroughly characterized two mouse LUSC cell models, i.e. cell lines UN-SCC679 and UN-SCC680 derived from the NTCU-induced mouse model in A/J mice. UN-SCC679 and UN-SCC680 cells carried different somatic mutations and gene expression profiles, with tumor mutation burdens comparable to those of LUSC patients. UN-SCC680 cells responded partially to treatment with anti-PD-1 antibody, whereas UN-SCC679 cells were refractory to the treatment. The immune landscape study revealed a cold IS of both cell lines, with a more-immunosuppressive tumor microenvironment in UN-SCC679 tumors. Finally, both cell lines demonstrated metastatic potential with the ability to grow metastatic lesions in the brain, bones, liver and adrenal glands of different degree of aggressiveness. In summary, we believe we have generated a very valuable tool for further LUSC research, comprising two cell lines with complementary traits that recapitulate the complexity of LUSC in humans.Seven-week-old A/JOlaHsd mice (hereafter referred to as A/J mice) were obtained from Harlan Laboratories (Harlan-Winkelmann). All animal experiments were conducted in accordance with the standards set by the Declaration of Helsinki. The protocols were approved by the institutional animal care committee of our institution , following the legal and ethical requirements demanded by the European Communities Council Directive 2010/63/UE and according to the regulations of the Spanish Royal Decree 53/2003, which were drawn up to ensure that pain and suffering would be limited to the lowest level.−/−IL2Rg−/− immunodeficient mice and engrafted into syngeneic 8-week-old A/J mice as previously described . Both cell lines were cultured in RPMI 1640 supplemented with 10% Fetalclone serum (Thermo Fisher Scientific) and 100 U/ml penicillin-100 µg/ml streptomycin (Thermo Fisher Scientific). All cells were grown in a humidified incubator under 5% CO2 at 37°C. Cell lines were routinely tested for mycoplasma.LUSC tumors were induced by N-nitroso-tris-chloroethylurea  treatment applying 0.04M NTCU by skin painting twice a week for 20 weeks to 8-week-old A/J mice. Lungs of euthanized mice were excised, and tumor cell lines UN-SCC679 and UN-SCC680 were derived and cultured for 25 passes to ensure they were immortalized. Cells were then subcutaneously injected into the flanks of 6-week-old female Rag2escribed  , catalog no.: AM071-10M, 1:50, TE pH 9.0, 20 min at 95°C; TTF1 (Dako) catalog no.: M357529-2, 1:40, TE pH 9.0, 6 min at 98°C; P40  catalog no.: 3066, 1:200, TE pH 9.0, 20 min at 95°C; P63 (Abcam) cataloge no.: ab735, 1:100, EDTA pH 8.0, 20 min at 95°C. All primary antibody incubations were carried out overnight at 4°C. After detection of primary antibodies was performed with EnVision System (Dako), slides were counterstained with hematoxylin, dehydrated, and covered with DPX mounting medium.Whole-exome sequencing was performed on extracted DNA from the tumor-derived cell lines. Enrichment for sequencing was performed using the SureSelectXT Mouse All Exon kit (Agilent) following the manufacturer's protocol. Exon-enriched libraries were subjected to paired-end sequencing, 2×100 base pairs read length and 60× minimum coverage.https://uni-koeln.sciebo.de/s/JJdOLcanGNN7U92.Raw sequencing reads were aligned to the respective mouse reference genome (NCBI37/mm9). Alignment was performed with the BWA aligner. Concordant read-pairs were identified as potential PCR duplicates and subsequently masked in the alignment file. Somatic mutations and copy number alterations were determined using our in-house analysis pipeline . Data arGSE185054.Samples were prepared with the Illumina TruSeq Stranded mRNA kit following the manufacturer's instructions and sequenced as reverse paired-end (50 bp) runs on the Nextseq sequencer. For RNA sequencing analysis, raw fastq files were trimmed with Trimmomatic/0.36. Pseudoalignment was carried out to the mm10 reference genome and gene level counts were determined with Kallisto . Differein vivo tumor growth studies, 2×106 UN-SCC679 or UN-SCC680 cells were subcutaneously inoculated in both flanks of 8-week-old female Rag−/− IL2Rg−/− mice (Harlan-Winkelmann). Tumor volume (weight2×length×0.52) was measured with a digital caliper twice a week until sacrifice.Cell proliferation in 2D was assessed using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay, MTS (Promega). Experiments were carried out on the days indicated, according to the manufacturer's instructions and normalized to day 1 post seeding. For 6 UN-SCC679 or UN-SCC680 cells were subcutaneously inoculated in one flank of 8-week-old female A/J mice (Harlan-Winkelmann). When tumors reached a volume of 75 mm3, mice were randomized (n=6 per group) and treated intraperitoneally with three doses of 100 µg of the following antibodies: anti-PD-1 , anti-CTLA4  or PBS every 3 days from day 7 post inoculation. Tumor volume (weight2×length×0.52) was measured with a digital caliper twice a week until euthanasia.2×106 UN-SCC679 or UN-SCC680 cells were subcutaneously inoculated in one flank of 6-8-week-old female A/J mice (Harlan-Winkelmann). Thirteen days after cell inoculation, tumors were collected and processed for flow cytometry analysis as previously described  by using the murine-specific kit (NEL840001KT) from Akoya Biosciences  following the manufacturer's instructions. Extra Alexa Fluor tyramides Opal 540 (FP1494001KT), 620 (FP1495001KT) and 650 (FP1496001KT) from Akoya Biosciences were used as well. Citrate buffer pH6 was used for all antigen retrievals and removal of primary-secondary complexes formed with previous antibodies. Primary antibodies and its corresponding Opal reagents were as follows: FOXP3  at 1:600 dilution and combined with Opal 540 fluorophore; CD4 (Abcam ab183685), 1:400 dilution, Opal 520; CD8 , 1:500 dilution, Opal 620; and F4/80 , 1:400 dilution, Opal 650. Acquisition of multispectral images and unmixing were conducted with the Vectra Polaris Automated Quantitative Pathology Imaging System, using the Phenochart and InForm softwares .Multispectral analysis was performed using QuPath 0.3.0 software . Tumor r5 cells as detailed by in vivo system . For imaging analysis, M3 Vision software (v 1.1) was used. Photon flux was calculated for each mouse by using a circular region of interest for the whole body and extracted as photons/s/cm2/sr. All in vivo experiments were normalized to the luciferase signal at day 0.Cells had previously been transfected with a triple modality construct expressing GFP, luciferase and thymidine kinase . Eight-wt-test. Non-normal samples were analyzed using the Mann–Whitney test  or the Median test .Sample size was chosen based on similar experiments previously published by the authors. For comparison of two groups, sample normality and variance were explored . Groups with normal distribution followed a two-tailed


O-propargylated mollugin with aryl azides. All the compounds were evaluated for their cytotoxicity on five human cancer cell lines  using MTS assays. Among the synthesized series, most of them showed cytotoxicity and most of all, compounds 14 and 17 exhibited significant cytotoxicity of all five cancer cell lines.A new series of mollugin-1,2,3-triazole derivatives were synthesized using a copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of corresponding  Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) . Briefly, cells were seeded into each well of a 96-well cell culture plate. After 12 h of incubation at 37 °C, the test compound (40 μM) was added. After incubated for 48 h, cells were subjected to the MTS assay. Compounds with a growth inhibition rate of 50% were further evaluated at concentrations of 0.064, 0.32, 1.6, 8, and 40 μM in triplicate, with cisplatin and paclitaxel (MeilunBio) as positive controls. After the incubation, MTS (20 μL) was added to each well and the incubation continued for 4 h at 37 °C. After sufficient reaction, the light absorption value of each well was read by Multiskan FC at 492 nm. The IC50 value of each compound was calculated with Reed and Muench’s method.The following human cancer cell lines were used: HL-60, A-549, SMMC-7721, MCF-7, and SW-480. These cells were obtained from American type culture collection (ATCC) . All the cells were cultured in RPMI-1640 or Dulbecco’s modified Eagle medium (DMEM) medium , supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere with 5% CO14 and 17 exhibited cytotoxicity of all five cancer cell lines significantly and compound 36 could enhance the cytotoxicity of lung cancer cells (A549) specifically. Structure and activity relationship (SAR) analysis reveals that electron-donating groups including hydroxyl, methoxy, and alcohol hydroxyl groups are essential for retaining the cytotoxicity to derivatives. In addition, for derivatives containing methoxy groups that the cytotoxicity may increase with the number of methoxy groups. Based on the SAR studies, we believe that the enhancement of cytotoxicity of the derivatives may be caused by the aromatic ring becoming electron-rich or the electron-donating atoms with lone pairs provided by electron-donating groups available to serve as hydrogen bond acceptors with the active site, which is worthy of further study.In conclusion, 40 1-substituted 1,2,3-triazole-mollugin derivatives were synthesized through Huisgen 1,3-dipolar cycloaddition reaction and evaluated for cytotoxicity against a series of five different human cancer cell lines  along with the parent molecule. Most of the derivatives showed better cytotoxicity than parent molecule. It is worth mentioning that our experiment results showed that compound


Most tested compounds induced cell death through apoptosis and necrosis. There was no correlation established between beta adrenoblocker anticancer activity and their selectivity to beta adrenoreceptors.Beta adrenoblockers are a large class of drugs used to treat cardiovascular diseases, migraines, glaucoma and hyperthyroidism. Over the last couple of decades, the anticancer effects of these compounds have been extensively studied. However, the exact mechanism is still not known, and more detailed studies are required. The aim of our study was to evaluate the anticancer activity of beta adrenoblockers in non-small cell lung cancer cell lines A549 and H1299. In order to find the relationship with their selectivity to beta adrenoreceptors, selective  and non-selective  beta blockers were tested. The effect on cell viability was evaluated by MTT assay, and the activity on cell ability to form colonies was tested by clonogenic assay. The type of cell death was evaluated by cell double staining with Hoechst 33342 and Propidium iodide. The most active adrenoblockers against both tested cancer cell lines were propranolol and betaxolol. They completely inhibited lung cancer cell colony formation at 90% of the EC Lung cancer is the most common type of cancer and a leading cause of death worldwide, accounting for an estimated 9.6 million deaths in 2018 .The catecholamines norepinephrine and epinephrine, also called noradrenaline and adrenaline, are neurotransmitters that tare simultaneously released from the sympathetic nervous system and adrenal gland as a response to physiological and psychological stress, otherwise called flight-or-fight response. They regulate the activity of organs and cells related to stimulation of the sympathetic nerve system. According to scientists, the elevated concentration of catecholamines promotes growth of lung adenocarcinoma micro metastasis . ConversBeta adrenoblockers are a large class of drugs mainly used to manage cardiovascular diseases, migraines, glaucoma and hyperthyroidism. Over the last couples of decades, the anticancer effects of these compounds have been extensively studied. The first evidence about beta adrenoreceptor involvement in lung cancer development occurred in 1989 . AccordiAccording to the results of clinical trials, beta adrenoblockers increase the survival rate of patients suffering from breast, prostate, ovarian, colorectal, skin and lung cancer. In recent years, new evidence has shown that overall survival of patients, who received beta adrenoblockers combined with radiotherapy, increased by 22% compared to the control group . DespiteAlthough the anticancer activity of beta adrenoblockers has been observed for almost two decades, no unified anticancer mechanism of action was discovered. Considering the problem and prevalence of lung cancer treatment, we decided to investigate the anticancer activity of beta adrenoblockers in the NSCLC lines A549 and H1299. These cell lines are derived from lung adenocarcinomas, but they represent different cell types. A549 cell line is a type II alveolar epithelium of characteristic morphology and functions ,23, and In this work, the beta adrenoblockers’ effect on cell viability, clonogenicity and the type of cell death was investigated. As the model compounds for our study, we chose the first-generation beta adrenoblockers acting as antagonists on both beta-1 and beta-2 adrenoreceptors . Also, the second-generation beta adrenoblockers, which bind stronger to the beta-1 receptors, were selected . Pindolol also has partial agonist activity ,28. We a50 values of both most active compounds, propranolol showed a stronger effect on viability of the H1299 cell line, while betaxolol acted in the same way in both cell lines (p < 0.05) (All tested compounds reduced NSCLC cell viability at the highest used concentration of 500 µM. Propranolol and betaxolol were the most active compounds in both cell lines a. After  < 0.05) b.50 values were 119.3 ± 12.7 µM and 98.8 ± 10.3 µM in A549 and H1299 cell lines, respectively). Betaxolol activity was about twice lower compared to propranolol .Propranolol possessed the highest antiproliferative activity  (The tested beta adrenoblockers showed a different effect on NCLSC cell colony formation . Propran < 0.05) a,b. All Slightly weaker than propranolol and betaxolol, the number and area of A549 cell colonies was reduced by metoprolol at the higher used concentration in this study. The non-selective beta blockers timolol and pindolol and the selective beta blocker esmolol were found to possess a similar activity between them. Also, these non-selective beta blockers were less active compared to propranolol and betaxolol. The lowest inhibition of A549 cell colony formation ability was established for the selective beta blocker atenolol.p < 0.05). The most active compounds were propranolol and betaxolol, while esmolol and metoprolol showed slightly lower activity (p < 0.05).Similar trends of the effect of beta blockers on the H1299 cell line have been identified in the study. All compounds except atenolol had a statistically significant reduction in the number of colonies and the area occupied by these cells  (p > 0.05).Only betaxolol at a concentration of 10% of EC < 0.05) c,d. NoneMost tested compounds induced cell death through apoptosis and necrosis. In A549 cell lines, apoptosis was mainly induced, while in H1299 cell line compounds induced both apoptosis and necrosis .50 value (p < 0.05) . Only atenolol at a lower concentration did not induce apoptosis in the H1299 cell line (p > 0.05)  a. No sta > 0.05) b.No statistically significant difference was found between the beta adrenoblockers’ effect on cell apoptosis between cell lines.p < 0.05) (p < 0.05).Beta adrenoblockers mainly induced necrosis in H1299, but not in the A549 cell line c. All of < 0.05) d. Metopr50 values can be explained by the fact that the expression of receptors varies between different types of cell lines. In general, it is thought that non-selective beta adrenoblockers have a stronger effect on cell viability than that of beta-1 selective compounds [The effect of beta adrenoblockers on cell viability is a common subject of different scientific studies. Propranolol after 72 h of incubation inhibited cell viability of lung cancer A375 and melanoma P8 cell lines at concentrations 77.30 and 60.30 μM, respectively . Similarp < 0.05), while betaxolol activity was the same in both cell lines (p > 0.05). However, both NSCLC cell lines, A549 and H1299, possess the K-Ras gene mutation that is thought to be responsible for lower sensitivity to non-selective beta adrenoblockers [We found that the antiproliferative activity of beta adrenoblockers is not correlating with their selectivity to the receptors and might be dependent on the compound lipophilicity and membrane stabilizing activity. Beta-2 adrenoreceptors in lung adenocarcinoma are responsible for lymphatic permeation and vascular invasion . Howeverblockers . In addiblockers .The effect of beta adrenoblockers on colony formation is not a common subject of scientific research. Min discovered that propranolol and atenolol at 10 µM concentrations suppresses the growth and ability of A549 and H0CC-15 cells, treated by NNK, to form colonies . In thisZhang concluded that a 100 μM concentration of metoprolol does not cause apoptosis in pancreatic cell lines . In thisIn another study, propranolol at 50 μM concentration did not cause apoptosis of gastric adenocarcinoma in the BGC-823 and SGC-7901cell lines, but in combination with radiotherapy after 48 h incubation it induced apoptosis, clonogenic survivability and cell viability . In our In order to evaluate impact of beta adrenergic receptors on the type of cell death, the effect of the beta-2 selective adrenoblocker butoxamine, non-selective propranolol and beta-1 selective metoprolol were used to induce apoptosis in a PC-2 pancreatic cancer cell line . The apoAtenolol (99% pure), betaxolol (96% pure), esmolol (98% pure), timolol (99% pure) and pindolol (99% pure) were purchased from Abcam , metoprolol (98% pure) was purchased from Alfa Aesar , and propranolol (99% pure) was purchased from Acros Organic . All tested compounds were dissolved in dimethylsulfoxide  which was obtained from Sigma-Aldrich Co. .TrypLE Express, Dulbecco’s modified Eagle high glucose medium (DMEM GlutaMAX), fetal bovine serum (FBS), penicillin/streptomycin solution , and phosphate buffered saline  were purchased from Gibco . The aqueous 16% paraformaldehyde solution (PFA), the Hoechst 33342 (1 mg/mL) solution, and the Propidium iodide (1 mg/mL) solution were obtained from Thermo Fisher Scientific .3--2,5-diphenyltetrazolium bromide  and crystal violet (≥90%) were purchased from Sigma-Aldrich Co. . Ethanol (96.6%) was obtained from Stumbras, LLC .All cell culture plastic ware was purchased from Thermo Fisher Scientific, Corning  and Techno Plastic Products .2. All cell cultures routinely were grown to 70% confluence and trypsinized with 0.125% TrypLE™ Express solution  before passage. They were used until passage 20.Human NSCLC cell lines A549 and H1299 were obtained from the American Type Culture Collection . Both cell lines were cultured in Dulbecco’s Modified Eagle’s Medium GlutaMAX , supplemented with 10% FBS and 1% antibiotics. Cells were incubated at 37 °C temperature in a humidified atmosphere containing 5% COCell viability was evaluated by MTT assay, as described elsewhere . BrieflyAfter 72 h, 20 µL of MTT 0.5 mg/mL solution was added into each well of a 96-well plate, and cells were incubated at 37 °C for 3 h. Next, the supernatant was removed and the formed formazan crystals were dissolved in 100 µL of DMSO. The absorbance was measured at 570 nm and 630 nm reference wavelengths using a multi-detection microplate reader. Experiments were repeated three times independently and the results were presented as means ± SD.50) values, reducing cell viability by 50%, were calculated.Applying Hill fit to compound dose—cell metabolic activity (absorbance) curves, the half maximal effective concentration  and analysed using Genesys software . The number and percentage area of colonies were calculated.The compound effect on cell colony formation was tested by clonogenic assay as described elsewhere . Briefly2. Next, either 10 or 90% of EC50 values of adrenoblockers were added to the wells. After 72 h, 3 µL of aqueous solution of Hoechst 33342 (1 mg/mL) and 1 µL of aqueous solution of Propidium iodide (1 mg/mL) were added to each well. After 10 min, images of cells were taken by an inverted fluorescent microscope . Apoptotic and necrotic cells were counted, and the percentage number of cells was calculated.Lung cancer cells were seeded in 24-well plates at a concentration 15,000 cells/well and incubated for 24 h at 37 °C in an atmosphere containing 5% COHoescht 33342 is a cell permeable dye and binds to the DNA in cells. Therefore, the stained nuclei of vital cells emit blue-cyan fluorescent light and are visible as blue colored. Apoptotic cells display condensed DNA and fragmented nuclei in blue. Propidium iodide is not cell permeable and binds to the double-stranded DNA of cells where the plasma membrane has been compromised. Non-viable, necrotic cells are seen as red colored. Propidium iodide does not stain live or early apoptotic cells due to the presence of an intact plasma membrane .t-test was used and p-values were calculated. A value of p < 0.05 was considered as the level of significance.A statistical analysis was performed using Microsoft Office Excel 2007 software , evaluating an average and standard deviation of at least three measurements. A Student’s 50 values were 251.3 ± 14.6 µM and 252.2 ± 7.6 µM in A549 and H1299 cell lines, respectively). Therefore, it could be concluded that the cytotoxicity of beta adrenoblockers against the tested lung cancer cell lines is not dependent on their selectivity on beta adrenoreceptors.Our results show that both selective and non-selective beta adrenoblockers, especially betaxolol and propranolol, reduce the viability of the NSCLC cell lines H1299 and A549. Propranolol, which is a non-selective beta adrenoblocker, showed the strongest effect on H1299 cell viability, while the selective agent betaxolol possessed a similar activity against both tested cell lines. Betaxolol activity was about twice lower compared to propranolol (ECOverall, it has been established that betablockers inhibit the formation of cell colonies and induce apoptosis and necrosis. No statistically significant difference was found between the beta adrenoblocker effect on cell apoptosis in both NSCLC cell lines. In A549 cell lines, apoptosis was mainly induced, while the H1299 cell line compounds induced both apoptosis and necrosis.The anticancer activity of the tested beta adrenoblockers is not related to the selectivity to beta adrenoreceptors. Therefore propranolol and betaxolol showed the strongest anticancer activity in vitro, and both compounds are worthy of further investigation and could be considered as therapeutic alternatives with regard to solving chemoresistance.


In endoscopic core ESCC biopsies taken from 61 patients who underwent neoadjuvant chemotherapy (NAC) (5‐fluorouracil and cisplatin), low MRE11A and high UBQLN4 protein levels were associated with reduced pathological response to NAC . Multivariable analysis of surgically resected ESCC tissues from 59 patients revealed low MRE11A and high UBLQN4 expression as independent factors that can predict shorter overall survival . Suppression of MRE11A expression was associated with cisplatin resistance in ESCC cell lines. Additionally, MRE11A was found to be ubiquitinated after cisplatin treatment. We observed an amplification of UBQLN4 gene copy numbers and an increase in UBQLN4 protein levels in ESCC tissues. Binding of UBQLN4 to ubiquitinated‐MRE11A increased MRE11A degradation, thereby regulating MRE11A protein levels following DNA damage and promoting cisplatin resistance. In summary, MRE11A and UBQLN4 protein levels can serve as predictors for NAC response and as prognostic markers in ESCC patients.Resistance to standard cisplatin‐based chemotherapies leads to worse survival outcomes for patients with esophageal squamous cell carcinoma (ESCC). Therefore, there is an urgent need to understand the aberrant mechanisms driving resistance in ESCC tumors. We hypothesized that ubiquilin‐4 (UBQLN4), a protein that targets ubiquitinated proteins to the proteasome, regulates the expression of Meiotic Recombination 11 Homolog A (MRE11A), a critical component of the MRN complex and DNA damage repair pathways. Initially, immunohistochemistry analysis was conducted in specimens from patients with ESCC  and demonstrated their clinical utility. Briefly, we showed that cisplatin treatment promotes Meiotic Recombination 11 Homolog A (MRE11A) ubiquitination. Binding of ubiquilin‐4 (UBQLN4) to ubiquitinated‐MRE11A increased MRE11A degradation, thereby promoting cisplatin resistance. Both MRE11A and UBQLN4 can predict neoadjuvant chemotherapy response and serve as prognostic markers in ESCC patients. AbantibodyAJCCAmerican Joint Committee on CancerCo‐IPCo‐ImmunoprecipitationDSBdouble‐strand breakEACesophageal adenocarcinomaESCAesophageal carcinomaESCCesophageal squamous cell carcinomaEVempty vectorFFPEformalin‐fixed paraffin‐embeddedIFimmunofluorescenceIHCimmunohistochemistrymAbmonoclonal antibodyMRE11AMeiotic Recombination 11 Homolog AMRNMRE11‐RAD50‐NBS1NACneoadjuvant chemotherapyNSnot significantOSoverall survivalOVoverexpressionpAbpolyclonal antibodySDstandard deviationTCGAThe Cancer Genome AtlasUBAubiquitin‐associated domainUblubiquitin‐like domainUBQLN4ubiquilin‐41Esophageal cancer represents the 7th cause of cancer death globally . The twoThe MRE11‐RAD50‐NBS1 (MRN) complex has an important role in DDR and chemotherapy treatment. MRN complex sensors and orchestrates DDR in double‐strand breaks (DSB), stalled replication forks, dysfunctional telomeres, and immune responses , 18. AltUbiquilin family proteins function as adaptors between ubiquitinated proteins and the proteasome , 28, 29.MRE11A gene are rare in ESCC tumors. UBQLN4 has a role in controlling nuclear protein levels by targeting ubiquitinated proteins to the proteasome  ESCC patients who underwent NAC followed by surgery at Keio University Hospital, Department of Surgery, Tokyo, Japan, between 2011 and 2016. These patients did not undergo adjuvant treatment. Specimens met the following criteria: (a) histologically proven ESCC, (b) no previous history of chemotherapy or chemoradiotherapy for any malignancies, and (c) no microscopic residual tumor (R0). For NAC, cisplatin plus 5‐fluorouracil was repeated twice every 3 weeks (day 1: cisplatin by 80 mg·m mg·m−2) . Tumor rH‐score values. Univariate and multivariate analysis was performed to determine associations between H‐scores and clinical variables.Formalin‐fixed paraffin‐embedded tissues from 59 surgically resected ESCC primary tumors were obtained from patients who underwent up‐front surgery at Keio University Hospital from 1997 to 2002 . IHC forAll of the experiments followed World Medical Association Declaration of Helsinki and the NIH Belmont Report principles. Tissue specimens were coded according to HIPAA recommendation. This study was approved by Keio University School of Medicine Ethics Committee with a registration number of 2016‐0233 and 2016‐241. All human samples and clinical information for this study were obtained according to the protocol guidelines approved by the Joint Institutional Review Board of Providence Saint John's Health Center/ Saint John's Cancer Institute and the Western Institutional Review Board (USA).2.22. All human cell lines have been authenticated using short tandem repeat profiling within the last 3 years. All experiments were performed with mycoplasma‐free cell lines.Established human ESCC cell lines TE‐4 (CVCL_3337), TE‐8 (CVCL_1766), and TE‐10 (CVCL_1760) were obtained from Keio University. Cell lines were cultured in Dulbecco's modified Eagle's medium or RPMI supplemented with 10% FBS at 37 °C with 5% CO2.3−1.MRE11A EX‐q‐0491‐M35 and the empty control vector EX‐EGFP‐M35 were from Genecopoeia, Rockville, MD, USA; ubiquitin B pCMV6 vector and the empty vector (EV) were from Origene, Rockville, MD, USA; UBQLN4 was cloned in pLNCX2 plasmid as described previously . For stam of cisplatin  supplemented medium for 3 days. Afterward, the medium was replaced and the cell lines were cultured until the resistant clones proliferated; the treatment was repeated with 3 μm and then 5 μm of cisplatin supplemented medium. Then, the cell lines were continuously cultured in 2.5 μm of cisplatin for 4 weeks. Cell lines were recovered in cisplatin‐free medium and used for further assays. For knockdown experiments, TE‐4 cell lines were transfected with 20 nm pool siRNA UBQLN4 or nontargeting Pool  using jetPRIME Polyplus transfection . TE‐8 and TE‐10 cell lines were transfected with 50 nm pool siRNA MRE11A or nontargeting Pool  using jetPRIME Polyplus transfection.For establishment of cisplatin‐resistant ESCC cell lines, TE‐8 and TE‐10 cell lines were cultured in 2 μ2.4mantra snap Software 1.03 . H‐scores were calculated using the inform 2.4 software (Perkin Elmer) and following manual instructions. Briefly, the tumor area was segmented from the stromal area and nuclei/cytoplasm compartments were distinguished by detecting the intensity of hematoxylin and/or 3,3'‐diaminobenzidine (DAB) staining. The optical signal threshold to classify the score into 4‐bins was set to 0.05, 0.12, and 0.2. Five slides per case were analyzed and the average H‐score was then used as the final value. The cutoff values for UBQLN4 and MRE11A were determined by considering H‐score values for the mean value observed in normal adjacent epithelia esophagus tissues plus 10 SD.Endoscopic biopsy specimens and surgically resected ESCC tumors were stained with UBQLN4 monoclonal antibody  and/or MRE11A polyclonal antibody  as previously described . Images 2.5m MG‐132  and/or 5 μm cisplatin for 12 h. After incubation, cell lines were washed and lysed in Co‐Immunoprecitation (Co‐IP) buffer  by gently pipetting. The whole‐cell (WC) lysates were quantified by bicinchoninic acid assay, and 250 μg of WC lysate  was incubated with UBQLN4 mAb  and mouse (G3A1) mAb control ; or UBQLN4‐Ps318 pAb  and mouse (G3A1) mAb control ; or MRE11A pAb  and normal rabbit pAb control ; or DDDDK tag pAb and normal rabbit pAb control  in Co‐IP buffer. Immunocomplex was further incubated with Dynabeads™ Protein A  for 1 h at 4 °C. Only for the experiment showed in Fig. m of recombinant human USP2 catalytic domain protein  for 30 min at 37 °C, as indicated. Beads were washed three times with Co‐IP buffer, recovered, and boiled in Fluorescent Master Mix for 5 min at 95 °C in a dry bath. After centrifugation, the supernatants (SN) were recovered and protein concentration was adjusted to 1 μg·μL−1 with 1× MM reagent. Samples were analyzed by automated western blot system [Cell lines were transfected with MRE11A and UBB plasmids as indicated in each experiment. After 24 h, cell lines were treated with 5 μt system , 34.2.67/mL. The sorting microfluidic chip was precoated with On‐chip T‐buffer before loading the sample. After priming the chip, excessive T‐buffer was removed from all reservoirs, and replaced with fresh culture medium. Then, the prepared samples were loaded. Gates were set and the samples were sorted according to the cell size and GFP positivity of interest. The sorted samples were retrieved, and the sorting process was repeated three times to increase the purity of the sorted cell lines. After sorting, cell lines were immediately cultured until 80% confluence and then frozen.The microfluidic chip‐based cell sorter, On‐chip Sort , equipped with blue (488 nm), violet (405 nm), and red (637 nm) lasers, was used according to the manufacturer's protocol. TE‐8 and TE‐10 UBQLN4‐OV cell lines were harvested, centrifuged, and diluted to 1 × 102.74) were seeded in 8‐well Falcon™ chambered culture slides (Fisher Scientific) and incubated for 48 h, followed by treatment with 5 μm cisplatin for 12 h. After that, cell lines were fixed and the protocol was followed as described in [imagej software .Cell lines (1–1.5 × 10ribed in  except t2.83) were seeded in a 96‐well plate. The number of viable cells was assessed using a Cell Titer‐Glo Luminescent  according to the manufacturers' instructions. For colony formation, ESCC cell lines (2 × 103) were seeded in a 6‐well plate and after 12 days of incubation processed as previously described [ESCC cell lines  with or without 5 μm MG‐132 at different time points . After incubation times, cell lines were harvested and processed for protein extraction. Western blot for MRE11A and β‐actin was performed and their expression was quantified over time and adjusted to β‐actin levels [TE‐10 cell lines (80% confluency) were incubated with 200 μg·mLn levels , 34, 35.2.106 cells/dish) were cultured in 100‐mm dishes and harvested with 3 mL cold PBS/phosphatase inhibitor buffer. Cells were centrifuged and the WC pellet was gently suspended in 500 µL 1× hypotonic buffer and incubated for 15 min on ice. Then, 25 µL of detergent was added to induce cell lysis. After cell lysis, the cytoplasmic fraction (supernatant) was separated from the nuclear fraction (pellet) by centrifugation (for 30 s at 14 000 g). Then, the nuclear fraction (pellet) was resuspended in 50 µL of the complete lysis buffer and incubated with 2.5 µL detergent for 30 min on ice. The nuclear lysates were centrifuged for 10 min at 14 000 g. The nuclear fraction (supernatant) was then collected. Both nuclear and cytoplasmic fractions were analyzed by automated western blot.Nuclear and cytoplasmic fractions were isolated from the TE‐10 and TE‐8 cell line with the Nuclear Extract Kit . Cells  and the protein concentration was adjusted to 0.5–1 μg·μL−1. The results were analyzed using the Compass Software (Protein Simple) and imagej software . The primary Ab dilutions are shown in Table Automated western blot was performed according to the manufacturer's protocol . Br2.12t‐test was used for normally distributed data, and the nonparametric Wilcoxon rank‐sum test was used for non‐normally distributed data. One‐way ANOVA test and Bonferroni post hoc test was applied in the quantification of IF assays. Two‐way ANOVA test and the Bonferroni post hoc test were applied for cell proliferation and drug sensitivity analysis. Correlation analyses were performed using Pearson's or Spearman's correlation coefficient (r) according to the data distribution. OS was compared using the Kaplan–Meier method and log‐rank test. Multivariable analysis was performed by the Cox proportional regression model to analyze the independent predictor for OS. Significant factors considering the covariate factors and age were included for the multivariable analysis. A two‐sided P < 0.05 was considered statistically significant: *P < 0.05; **P < 0.01; ***P < 0.001 and NS, not significant. All analyses were performed with graphpad prism 5  or r version 3.5.0 , and th33.1MRE11A expression was analyzed in the The Cancer Genome Atlas (TCGA) esophageal carcinoma (ESCA) dataset [n = 186) of which all 95 ESCCs were analyzed  in about 4% (4 of 95) and reduced in 15% (z‐score ≤ −1.5) of ESCC patients  clinically annotated  compared to primary ESCC tumors  had a worse prognosis compared to those with high MRE11A expression  = 5.11, 95% confidence interval (CI) 1.45–18.03, Table To further elucidate the clinical significance of MRE11A, IHC was performed and SCC Fig. A. PatienH‐score values demonstrated that MRE11A expression was significantly increased in responder (n = 8) compared to nonresponder ESCC patients who underwent NAC  in ESCC cell lines and analyzed them for cisplatin sensitivity. MRE11A knockdown promoted cisplatin resistance in ESCC cell lines Fig. . ConversIn order to determine whether MRE11A was associated with cisplatin resistance, we established two cisplatin‐resistant ESCC cell lines. Cisplatin resistance in ESCC cell lines was confirmed by drug sensitivity assays Fig. ,J. In ag3.3We first examined the expression of MRE11A and UBQLN4 protein levels in three ESCC cell lines . Western blot analysis showed that when ESCC cell lines have increased UBQLN4, they consistently have low MRE11A protein levels Fig. . EctopicOur hypothesis is that during DNA damage ubiquitinated‐MRE11A levels increased in the DNA damage sites and UBQLN4 may help in promoting the proteasome‐mediated degradation. Thus, we examined the interaction between MRE11A and UBQLN4 using Co‐IP assays. For Co‐IPs, the ESCC cell lines were treated with cisplatin (to enhance the levels of ubiquitinated‐MRE11A and promote the interaction with UBQLN4) and with MG‐132 inhibitor . As shown in Fig. Then, we performed experiments to assess the changes in ubiquitinated‐MRE11A after cisplatin‐induced DNA damage. To do this, we transfected TE‐10 UBQLN4‐OV cells with UBB‐DDK and used anti‐DDK antibody to immunoprecipitate endogenous tagged ubiquitinated‐MRE11A in cisplatin‐treated ESCC cell lines in the presence or absence of MG‐132. To demonstrate that ubiquitinated‐MRE11A was bound to the beads, we pretreated the immunocomplexes with buffer or the recombinant catalytic domain of the de‐ubiquitinase USP2. If the interaction would be mediated by ubiquitination, USP2 would reduce the binding of MRE11A in the immunocomplexes. Ubiquitinated‐MRE11A levels were increased in ESCC cell lines treated with cisplatin in the presence of MG‐132 compared to absence of MG‐132 Fig. A. MoreovUsing Co‐IPs, we assessed for ubiquitinated‐MRE11A binding to UBQLN4. To do that, we transfected TE‐10 UBQLN4‐OV cells with UBB‐DDK and used anti‐DDK antibody to co‐immunoprecipitate endogenous ubiquitinated‐MRE11A DDK‐tagged and UBQLN4 in cisplatin‐treated ESCC cell lines in the presence of MG‐132. Ubiquitinated‐MRE11A co‐immunoprecipitated with UBQLN4 in ESCC cell lines Fig. . These rIn order to analyze MRE11A protein stability in the presence of high UBQLN4 levels, cycloheximide chase assays were performed in UBQLN4‐OV cell lines and compared to respective control EV cell lines. A significant decrease was observed for MRE11A protein levels in UBQLN4‐OV compared to the control EV cell lines Fig. . In IF a3.4UBQLN4 was highly expressed (mRNA z‐score ≥ 1.5) in about 24% (23 of 95) and decreased in only 2% (mRNA z‐score ≤ 1.5) of ESCC patients  in this region in primary ESCC tumors using the TCGA ESCA dataset. Forty‐four (48.4%) of the primary ESCC tumors showed a gain in the copy number for the UBQLN4 gene  was significantly higher than normal adjacent esophageal epithelia  had a significantly worse prognosis compared to those with low UBQLN4  and high UBQLN4/low MRE11A (n = 10) showed that patients with high UBQLN4/low MRE11A have a worse prognosis  compared to the responder group (n = 8)  compared to si‐Ctrl‐treated cell lines Fig. . UBQLN4 3.6Since UBQLN4 expression determines resistance to cisplatin in ESCC cell lines, we assumed that UBQLN4 may reduce the DNA damage after cisplatin treatment. To determine the DNA damage, two markers (53BP1 and γ‐H2AX) were analyzed by IF in TE‐10 and TE‐8 EV and UBQLN4‐OV cell lines cisplatin‐treated or untreated. As anticipated, ESCC cell lines with UBQLN4‐OV presented a reduction in 53BP1 Fig.  and γ‐H24in vitro using two cisplatin‐resistant ESCC cell lines. In addition, low MRE11A expression in up‐front surgically resected ESCC tissues was related to significantly worse OS. It is accepted that the loss of MRN complex function results in DNA repair deficiency and increase sensitivity to DNA damaging therapies [This study showed that low MRE11A expression in tumor biopsies taken prior to NAC was associated with cisplatin resistance in ESCC, which was further validated herapies , 18, 22.herapies , 24, 25.herapies .in vitro results demonstrated that UBQLN4 OV alleviated DNA damage induced by cisplatin, while UBQLN4 knockdown caused the opposite effects.In ESCC patients, UBQLN4 expression was increased and associated with increased gene copy number. Moreover, UBQLN4 protein levels were found to be increased in core biopsies from ESCC patients who have poor response to NAC. Multivariable analysis showed that UBQLN4 was an independent prognostic factor to predict OS. These results suggest a potential role for UBQLN4 to predict cisplatin resistance in ESCC tumors. Additionally, our In order to identify molecular mechanisms regulating MRE11A expression at the DSB sites, we interrogated its relationship with UBQLN4. We demonstrated that DNA damage, induced by cisplatin, promotes UBQLN4 increase at DSB in ESCC cell lines. Furthermore, ubiquitinated‐MRE11A interacted with UBQLN4 during DNA damage induced by cisplatin, to be efficiently targeted to the proteasome for degradation. These results validated our previous observations demonstrating that UBQLN4 and ubiquitinated‐MRE11A accumulate at the DSB after inducing DNA damage in other solid tumors . In addiUBQLN2 and UBQLN4 have a conserved role as regulators of endoplasmic reticulum stress, autophagy, and mTOR signaling . In regaVarious attempts have been made to predict prognosis for patients with locally advanced ESCC after NAC, including diagnostic imaging , tumor m5UBQLN4 gene upregulates UBQLN4 expression in ESCC. Mechanistically, cisplatin treatment increased ubiquitinated‐MRE11A, which is targeted to the proteasome by UBQLN4 and leads to cisplatin resistance in vitro. As both MRE11A and UBQLN4 expressions were associated with clinical outcomes, they could serve as predictors for cisplatin‐based NAC response and survival outcomes in patients with primary ESCC tumors.Decreased MRE11A expression is associated with cisplatin resistance in primary ESCC tumors and cell lines. On contrary, increased copy number for The authors declare no conflict of interest.H‐score), writing—original draft preparation, reviewing, and editing. YS involved in data acquisition, analysis and interpretation , reviewing and editing. TN involved in data acquisition, analysis and interpretation , reviewing and editing. S‐CC involved in basic statistical and clinical analyses , reviewing, and editing. RDJ involved in data interpretation, reviewing, and editing. SH involved in clinical data analysis, reviewing, and editing. SO involved in clinical data analysis, reviewing, and editing. YS involved in conceptualization and design, reviewing, and editing. HT involved in conceptualization and design, reviewing, and editing. YK involved in conceptualization and design, reviewing, and editing. DSBH involved in conceptualization and design, supervision, funding, writing, reviewing, and editing. MAB involved in conceptualization and design, supervision, methodology, data acquisition, and interpretation , writing original draft, reviewing, and editing. All authors have read and agreed to publish the current version of the manuscript.TM involved in conceptualization and design, methodology, data acquisition, and interpretation  comparing si‐Ctrl and si‐MRE11A (pool siRNA) in TE‐10 (A) and TE‐8 (B) cisplatin‐resistant (Cis‐Res) cell lines. C‐D. Drug sensitivity assays comparing si‐Ctrl or si‐MRE11A (pool siRNA) in TE‐10 (C) and TE‐8 (D) cisplatin‐resistant (Cis‐Res) cell lines treated with different cisplatin concentrations . E‐F. Western blot analysis for MRE11A, UBQLN4, and β‐actin (loading control) comparing EV and MRE11A‐OV in TE‐10 (E) or TE‐8 (F) cisplatin‐resistant (Cis‐Res) cell lines. G‐H. Drug sensitivity assays comparing cisplatin‐resistant (Cis‐Res) TE‐10 (G) or TE‐8 (H) cell lines with EV or MRE11A‐OV and treated with different cisplatin concentrations (***P < 0.001). Error bars represent the mean ± SD from n = 3 replicates. Statistical differences were tested using two‐way ANOVA test and post hoc Bonferroni test .Click here for additional data file.Fig. S3. Ubiquitinated‐MRE11A interacts with UBQLN4. A. IP assay in TE‐10 UBQLN4‐OV and UBB‐OV cell lines that were treated with cisplatin (5 μm) and MG‐132 (5 μm) using anti‐DDK IgG Ab or control IgG Ab. Before elution, the immunocomplexes were treated for 30 min at 37 °C with 50 nm of recombinant human catalytic domain of USP2 or buffer as indicated. MRE11A and ubiquitinated protein levels (ubiquitinated‐MRE11A) were assessed in whole‐cell (WC) lysates and IP fractions (IP). B. Co‐IP assay in TE‐10 UBQLN4‐OV and UBB‐OV cell lines that were treated with cisplatin (5 μm) and MG‐132 (5 μm) using anti‐MRE11A IgG Ab or control IgG Ab. UBQLN4, DDK tag, and MRE11A (loading control) protein levels were assessed in whole‐cell (WC) lysates and Co‐IP fractions (Co‐IP).Click here for additional data file.Fig. S4. UBQLN4 promotes MRE11A degradation in ESCC cell lines. A‐D. Immunofluorescence staining for MRE11A was performed in cisplatin‐treated  or untreated TE‐10 EV (A), TE‐10 UBQLN4‐OV (B), TE‐8 EV (C), and TE‐8 UBQLN4‐OV (D) cell lines. MRE11A (red), DAPI (blue), and the merged images are shown. Scale bars = 10 µm. E‐F. Quantification of MRE11A fluorescence intensity per cell in TE‐10 (E) and TE‐8 (F) cell lines . G‐H. Western blot for UBQLN4, MRE11A, and LSD1 (loading control) in the nuclear fractions isolated from TE‐10 (G) and TE‐8 (H) cell lines that were untreated or treated with cisplatin (5 μm). Error bars represent the mean ± SD from n = 3 replicates. Statistical differences were tested using ordinary one‐way ANOVA test and Bonferroni post hoc test (E and F).Click here for additional data file.Fig. S5. Staining patterns observed in IHC analysis for UBQLN4, A. Representative images are shown for the different IHC staining patterns observed in the ESCC surgical specimens. H‐scores = 0–50; 51–100; 101–150; 151–20; 201–250; 251–300. Scale bars = 50 µm. B. Correlation of nuclear and cytosolic staining H‐score values for UBQLN4 .Click here for additional data file.Fig. S6. Ubiquitinated‐MRE11A interacts with UBQLN4. A‐B. Immunofluorescence staining for γ‐H2AX was performed in cisplatin‐treated  or untreated TE‐4 si‐Ctrl (A) and si‐UBQLN4 (B) cell lines. γ‐H2AX (green), DAPI (blue), and the merged images are shown. Scale bars = 10 µm. C. Quantification of γ‐H2AX fluorescence intensity per cell in TE‐4 cell lines . D‐E. Immunofluorescence staining for γ‐H2AX was performed in cisplatin‐treated  or untreated TE‐8 si‐Ctrl (D) and si‐MRE11A (E) cell lines. γ‐H2AX (green), DAPI (blue), and the merged images are shown. Scale bars = 10 µm. F. Quantification of γ‐H2AX fluorescence intensity per cell in TE‐8 cell lines . G. Immunofluorescence staining for endogenous UBQLN4 and γ‐H2AX were performed in cisplatin‐treated  or untreated TE‐4 cell lines. UBQLN4 (red), γ‐H2AX (green), DAPI (blue), and the merged images are shown. Scale bars = 10 µm. H. Correlation between UBQLN4 and γ‐H2AX levels in cisplatin‐treated TE‐4 cell lines . Error bars represent the mean ± SD from n = 3 replicates. Statistical differences were tested using ordinary one‐way ANOVA test and Bonferroni post hoc test (C and F).Click here for additional data file.Fig. S7. Western blot uncropped images.Click here for additional data file.Fig. S8. Western blot uncropped images.Click here for additional data file.Table S1. List of antibodies (Ab) and dilutions utilized in this study.Click here for additional data file.