Radiotherapy plays an important role in the multimodal treatment of breast cancer. The response of a breast tumour to radiation depends not only on its innate radiosensitivity but also on tumour repopulation by cells that have developed radioresistance. Development of effective cancer treatments will require further molecular dissection of the processes that contribute to resistance.Radioresistant cell lines were established by exposing MDA-MB-231, MCF-7 and ZR-751 parental cells to increasing weekly doses of radiation. The development of radioresistance was evaluated through proliferation and colony formation assays. Phenotypic characterisation included migration and invasion assays and immunohistochemistry. Transcriptomic data were also generated for preliminary hypothesis generation involving pathway-focused analyses.Proliferation and colony formation assays confirmed radioresistance. Radioresistant cells exhibited enhanced migration and invasion, with evidence of epithelial-to-mesenchymal-transition. Significantly, acquisition of radioresistance in MCF-7 and ZR-751 cell lines resulted in a loss of expression of both ERα and PgR and an increase in EGFR expression; based on transcriptomic data they changed subtype classification from their parental luminal A to HER2-overexpressing (MCF-7 RR) and normal-like (ZR-751 RR) subtypes, indicating the extent of phenotypic changes and cellular plasticity involved in this process. Radioresistant cell lines derived from ER+ cells also showed a shift from ER to EGFR signalling pathways with increased MAPK and PI3K activity.This is the first study to date that extensively describes the development and characterisation of three novel radioresistant breast cancer cell lines through both genetic and phenotypic analysis. More changes were identified between parental cells and their radioresistant derivatives in the ER+ (MCF-7 and ZR-751) compared with the ER- cell line (MDA-MB-231) model; however, multiple and likely interrelated mechanisms were identified that may contribute to the development of acquired resistance to radiotherapy.The online version of this article (10.1186/s13014-019-1268-2) contains supplementary material, which is available to authorized users. Radiotherapy (RT) is a commonly used adjuvant treatment modality for a variety of solid cancers. Up to 83% of breast cancer patients benefit from radiotherapy either with curative or palliative intent . MultiplImproved understanding of the mechanisms underlying acquired radioresistance and the development of strategies to circumvent this clinical problem are required. Previous studies have shown that multiple factors are implicated in the development of radioresistance, including deregulated signalling pathways , oncogenic miRNA overproduction, enhanced DNA damage responses, the presence of cancer stem cells, epithelial-to-mesenchymal transition (EMT) and alterations in cancer metabolism, along with the effect of the tumour microenvironment itself (including hypoxia) . Many stIn comparison to the investigation of chemoresistance, the mechanisms underlying radioresistance development are poorly understood, partly due to a lack of radioresistant model systems. The use of global approaches to investigate resistance mechanisms is gaining interest, as this allows for the study of multiple pathways simultaneously and provides an overview of complex biological systems and response to treatment . In this− 1 penicillin and 50 mg ml− 1 streptomycin and incubated at 37 °C in a humidified atmosphere with 5% CO2. Cell lines obtained from the American Type Culture Collection  were authenticated by short tandem repeat (STR) profiling performed at Health England . All cell line DNA samples tested matched 9 of 9 tested core alleles in DNA from known cell line samples confirming their identity. All experiments were performed using cells, maintained at low passage number from these frozen stocks.Cell culture reagents were obtained from Gibco Thermo Fisher Scientific , unless otherwise stated. Human breast cancer cell lines ZR-751, MCF-7 and MDA-MB-231 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal calf serum (FCS), 50 U mlCells were irradiated using a Faxitron cabinet X-ray system 43855D . Radioresistant cell lines  were developed from their respective parental cell lines  by weekly exposure to single fractions of radiation. An initial dose of 2 Gy was followed by weekly incremental doses of 0.5 Gy for 12 weeks. During this period cells received a total radiation dose of 57 Gy. Cells were subsequently maintained with further weekly doses of 5 Gy.2O and, when dry, 50 μl SRB dye ) was added to each well and incubated for 30 min. Plates were washed 4 times in 1% glacial acetic acid and, when dry, 150 μl of 10 mM Tris-NaOH buffer (pH 10.5) was added to each well. The plates were incubated on a shaker for 60 min. Optical density was measured at 540 nm using a Biohit BP800 spectrophotometer  and Wallac 1420 Manager program . The half maximal inhibitory concentrations  were calculated using the GraphPad prism 7 package.Cells were seeded into 96 well plates (500 cells/well) and incubated for 24 h. Cells were drug-treated or exposed to radiation and fixed between 24 and 144 h after treatment by the addition of 50 μl cold 25% trichloracetic acid  per well at 4 °C for 1 h. Plates were washed in H3 cells/plate) and incubated for 24 h before radiation treatment. Once visible colonies had formed (approximately 50 cells per colony) in the untreated control group (approximately 10–14 days post-seeding) the plates were washed twice in PBS before fixing the cells with the addition of 5 ml of 1,9-dimethyl-methylene blue zinc chloride double salt . After 45 min the plates were washed and allowed to air dry before colonies were counted. Analysis was performed by calculating plating efficiencies and survival fractions for control and treatment plates . TAnalysis of transcriptomic data from ER+ parental and RR cell lines in respect of the WNT signalling pathway was investigated due to its reputed role in EMT and radioresistance . The WNT50: MCF-7 13.36 μM, MCF-7 RR 6.43 μM) and a significant reduction in migration, with a dose of 5 μM decreasing the migratory potential of the RR cell line to parental levels . Western blotting and IHC showed that the RR phenotype in the cell lines derived from ER+ cells was characterised by the loss of ERα and PgR expression, along with a gain in total EGFR expression  of 67 breast cancer cell lines. The MDA-MB-231 parental and RR cell lines, both treated and untreated, clustered tightly with each other in the dendrogram branch enriched for the basal breast cancer subtype. Both treated and untreated MCF-7 and ZR-75-1 parental cell lines were classified as luminal A whereas their RR derivates clustered independently and were enriched for normal-like (ZR-751 RR) and HER2-overexpressing (MCF-7 RR) intrinsic subtypes /protein kinase B (AKT) cascade is frequently overactive in a wide range of cancers and can trigger a cascade of responses related to cell survival and metabolism , 67. It In breast and other cancers radiation-induced proliferation has been linked with activation of EGFR and downstream components of the mitogen-activated protein kinase (MAPK) cascade, including phospholipase-C, Ras, and Raf-1 –73 and cThe use of multiple techniques ranging from gene to protein to functional experiments, which led to the validation of our results through independent means, represents one of the main strengths of this study. Additionally, the development of ER+ and ER- RR cell lines allowed for comparisons to be made between different molecular breast cancer subtypes. The aim of our study was to characterise these RR cell lines and identify pathways that could be related to radioresistance, rather than focusing on the reversal of radioresistance itself. However, following our successful characterisation, we now aim to use these cell lines in future studies to target these identified pathways and to identify biomarkers (genetic and secretomic signatures) that could be used for determining radiosensitivity and for the assessment of a tumour’s response to radiotherapy.In this study we developed a novel model to help improve the understanding and characterisation of radioresistant breast cancer cells and identified several important and likely interrelated networks contributing to the development of radioresistance. ER+ derived RR cell lines were characterised by a shift towards a more invasive mesenchymal phenotype with changes in oestrogen regulation, gain of EGFR signalling and change in subtype classification. The ER- cell line possessed these characteristics at the outset and hence its phenotype changed relatively little with the development of radioresistance. Here we have begun to dissect the mechanisms and signalling pathways involved in the development of radioresistance. Models of resistant disease as developed and examined in this study will be instrumental in future research and will aid the development of new therapeutic strategies for patients that either fail to respond or develop recurrent disease.Additional file 1:Table S1. RIN values for all samples used in gene expression analysis. (XLSX 10 kb)Additional file 2:Table S2. Overview of samples  used in each experiment or analysis. (XLSX 15 kb)Additional file 3:Figure S1. Colony formation assay comparing MCF-7, MCF-7 RR and MCF-7 rr (radioresistant cell line not radiated for 6 months (24 passages)) cell lines. (TIF 51 kb)Additional file 4:Figure S2. ICC and IHC staining of EMT markers in ZR-751 parental and RR cell lines. (TIF 2384 kb)Additional file 5:Figure S3. ICC and IHC staining of EMT markers in MCF-7 parental and RR cell lines. (TIF 2391 kb)Additional file 6:Table S3. Gene lists from a published EMT signature [ignature  and WNT ignature  applied Additional file 7:Figure S4. ICC and IHC staining for signalling receptors in ZR-751 parental and RR cell lines. (TIF 2818 kb)Additional file 8:Figure S5. ICC and IHC staining for signalling receptors in MCF-7 parental and RR cell lines. (TIF 2708 kb)Additional file 9:Figure S6. (A) SRB at 72 h and (B) Scratch assay at 24 h showing the effects of gefitinib on ZR-751 and ZR-751 RR cell lines . (TIF 170 kb)Additional file 10:Figure S7. Gene expression heatmap based on Pearson correlation hierarchical clustering with average linkage using a published list of intrinsic subtype genes [genefu R package Single Sample Predictor (SSP) algorithm [pe genes . Intrinslgorithm ; red=hig


Despite the clear involvement of V-ATPases in cancer, the therapeutic use of V-ATPase-targeting small molecules has not reached human clinical trials to date. Thus, V-ATPases are emerging as important targets for the identification of potential novel therapeutic agents. We identified a bisbenzimidazole derivative (V) as an initial hit from a similarity search using four known V-ATPase inhibitors (I–IV). Based on the initial hit (V), we designed and synthesized a focused set of novel bisbenzimidazole analogs (2a–e). All newly prepared compounds have been screened for selected human breast cancer  and ovarian cancer  cell lines, along with the normal breast epithelial cell line, MCF10A. The bisbenzimidazole derivative (2e) is active against all cell lines tested. Remarkably, it demonstrated high cytotoxicity against the triple-negative breast cancer (TNBC) cell line, MDA-MB-468 (IC50 = 0.04 ± 0.02 μM). Additionally, it has been shown to inhibit the V-ATPase pump that is mainly responsible for acidification. To the best of our knowledge the bisbenzimidazole pharmacophore has been identified as the first V-ATPase inhibitor in its class. These results strongly suggest that the compound 2e could be further developed as a potential anticancer V-ATPase inhibitor for breast cancer treatment.The vacuolar (H The V-ATPase proton pump is a macromolecular complex composed of at least 14 subunits organized into two functional domains, V1 and V0. The domain V1 is responsible for ATP hydrolysis and V0 provides the transmembrane proton channel imidazol]-2′-yl)phenol (compound 1) with appropriate substituted alkyl halides in the presence of cesium carbonate in dimethyl formamide (DMF) at reflux temperature (2a–e) using methanol and chloroform (3:7). We quickly developed a fast and efficient synthetic procedure to prepare novel bisbenzimidazole analogs using the one pot procedure. A general synthesis of bisbenzimidazole derivatives (perature . The cru+)-ATPase in breast as well as ovarian cancer cell lines -6-(1-methyl-4-piperazyl)benzimidazole trihydrochloride (compound 6-(4-Methylpiperazin-1-yl)-2'-(4-((1-methylpiperidin-4-yl)oxy)phenyl)-1H,3'H-2,5'-bibenzo[d]imidazole (2a) Yield: 12%, brown solid powder. m.p.: 165–166 °C; 1H-NMR  δ 2.06–2.18 , 2.55 , 2.70 , 2.89-3.00 , 3.19–3.28 , 4.74 , 7.06-7.09 , 7.18 , 7.53 , 7.72 , 7.97 , 8.09 , 8.29 ; MS (ESI): m/z 522.6 [M + H]+; HRMS (ESI): m/z calcd for C31H35N7O [M + H]+ 522.2981; obsd 522.2971.N,N-Dimethyl-3-(4-(6-(4-methylpiperazin-1-yl)-1H,3'H-imidazol]-2'-yl)phenoxy)propan-1-amine (2b). Yield: 15%, brown solid powder. m.p.: 171–172 °C; 1H-NMR  δ 1.90 , 2.20 2), 2.24 , 2.44 , 3.08–3.16 , 3.36–4.02 , 4.09 , 6.91–6.95 , 7.12 , 7.36–7.49 , 7.58–7.72 , 7.95–8.03 , 8.14 , 8.20–8.33 , 12.60 , 12.96 ; MS (ESI): m/z 510.6 [M + H]+; HRMS (ESI): m/z calcd for C30H35N7O [M + H]+ 510.2984; obsd 510.2983.N,N-Dimethyl-2-(4-(6-(4-methylpiperazin-1-yl)-1H,3'H-imidazol]-2'-yl)phenoxy)ethanamine (2c). Yield: 13%, yellow solid powder. m.p.: 197–198 °C; 1H-NMR  δ 2.44 3), 2.65 , 3.10–3.13 , 3.34–3.39 , 4.13 , 6.91–6.93 , 7.12 , 7.38–7.46 , 7.65 , 7.98 , 8.15 , 8.27 , 12.60 , 12.96 ; MS (ESI): m/z 496.4 [M + H]+; HRMS (ESI): m/z calcd for C29H33N7O [M + H]+ 496.2825; obsd 496.2820.2'-(4-(2-Methoxyethoxy)phenyl)-6-(4-methylpiperazin-1-yl)-1H,3'H-2,5'-bibenzo[d]imidazole (2d). Yield: 22%; yellow solid powder; m.p.: 208–209 °C; 1H-NMR  δ 2.35 , 2.62–2.65 , 3.16–3.18 , 3.39 , 3.68–3.70 , 4.05–4.08 , 6.98–7.00 , 7.07 , 7.46 , 7.52 , 7.57–7.58 , 7.95 , 8.16 ; 13C-NMR  δ 44.59, 50.25, 54.68, 57.85, 67.05, 70.63, 100.75, 114.57, 114.90, 120.98, 121.65, 124.17, 128.10, 134.5, 138.0, 148.06, 152.24, 153.70, 160.79, 171.56; MS (ESI): m/z 483.5 [M + H]+; HRMS (ESI): m/z calcd for C28H30N6O2 [M + H]+ 483.2508; obsd 483.2498.2'-phenyl)-6-(4-methylpiperazin-1-yl)-1H,3'H-2,5'-bibenzo[d]imidazole (2e). Yield: 16%; pale yellow solid powder; m.p.: 247–248 °C; 1H-NMR  δ 0.98 3), 1.70 , 2.24 , 3.12–3.30 , 4.11 , 6.91-6.96 , 7.13 , 7.58-7.72 , 7.28–7.49 , 7.94–8.02 , 8.13 , 8.20–8.35 , 12.58 , 12.94 ; 13C-NMR  δ 28.74, 29.23, 44.55, 46.94, 50.28, 54.68, 65.24, 100.10, 114.59, 114.97, 121.02, 121.32, 124.21, 128.14, 148.08, 152.32, 153.92, 161.14; MS (ESI): m/z 509.7 [M + H]+; HRMS (ESI): m/z calcd for C31H36N6O [M + H]+ 509.3029; obsd 509.3028.V), and bisbenzimidazole analogs 2a–e in the selected human breast cancer cell lines MDA-MB-231 , MDA-MB-468 , MCF-7  and the normal breast epithelial cell line, MCF10A, and the ovarian cancer cell lines, A2780, Cis-A2780, and PA-1 . Human breast cancer cell lines and the human ovarian cancer cell line (PA-1) were purchased from ATCC . The cisplatin-sensitive human ovarian carcinoma cell line, A2780, was obtained from Sigma-Aldrich. The cisplatin-resistant human ovarian cell line, Cis-A2780, was produced in our laboratory, as described in the literature [v/v) heat-inactivated fetal bovine serum , 100 U/mL penicillin G and 100 U/mL streptomycin . Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2 and were passaged by trypsinization after reaching 80–90% confluence.We studied the antiproliferative activity of an initial hit (compound terature . MDA-MB-3) were seeded into each well of 96-well plates and incubated overnight. Dimethyl Sulfoxide (DMSO) stock solutions of the compounds  were diluted in corresponding media and exposed to different concentrations for 48 h. All three independent experiments were performed in quadruplicates. Untreated cells were used as a negative control and data were presented as the mean ± standard deviation (S.D.). The absorbances at 492 nm were measured using a Microplate Reader . The compound concentration that inhibited cell growth by 50% of the untreated control (IC50) was calculated from the dose response curves constructed by plotting percentages of cell viability versus drug concentrations using nonlinear regression analysis by GraphPad Prism Software 7 .CellTiter 96 AQueous One Solution cell proliferation assay (MTS) ,30 was u6 cells per mL in the same buffer and placed on ice. H+ uptake was initiated by the addition of MDA-MB-231 cells  to pre-warmed HBSS (2.4 mL) containing AO  and placed in a cuvette. AO fluorescence was measured at an excitation wavelength of 495 nm and an emission wavelength of 540 nm using the LS-50B Luminescence Spectrometer . Nigericin  was added to collapse the pH gradient and unquench the AO fluorescence (See 1a and 1b). MDA-MB-231 cells  were treated with the standard V-ATPase inhibitor Baf A1 (1 μM), along with compounds V, 2d, and 2e (10 μM), for 20 min followed by addition of AO  for the proton pump activity . A decrease in the fluorescence intensities per min of Baf A1 and the potent compounds  was calculated and is expressed as a percentage of the control (mean ± SD of two independent experiments) (The proton pump activity was assessed by using the AO fluorescence quenching method . Bafilomriments) . V) as an initial hit compound. Synthesis and anticancer screening of a focused set of novel bisbenzimidazole derivatives indicated that they were potent against selected breast and ovarian cancer cell lines in vitro. The selected bisbenzimidazole analogs (2d and 2e) have demonstrated selective potency towards the TNBC cell line, MDA-MB-468. The compound 2e potently inhibited the proton pump activity, which is largely responsible for the acidification, and it showed selective anticancer activity. To the best of our knowledge, the bisbenzimidazole pharmacophore has been identified as the first V-ATPase inhibitor in its class. Together, our findings demonstrated that the bisbenzimidazole analog (2e) could be further developed as an anticancer vacuolar (H+)-ATPase inhibitor for the treatment of breast cancer.In conclusion, starting from the virtual screening practice using known small molecule V-ATPase inhibitors, we identified a bisbenzimidazole analog (


The prepared substances were screened for their in vitro antitumor activity on the osteosarcoma (HOS) and breast adenocarcinoma (MCF7) human cancer cell lines, where 2 showed moderate antitumor effect . The complex 2 was further tested on a panel of six others human cancer cell lines, including the malignant melanoma (G361), cervix carcinoma (HeLa), ovarian carcinoma (A2780), cisplatin-resistant ovarian carcinoma (A2780R), lung carcinoma (A549) and prostate adenocarcinoma (LNCaP). This substance was found to be moderate antitumor effective against G361 (IC50 = 17.3 μM), HeLa (IC50 = 31.8 μM) and A2780 (IC50 = 19.2 μM) cell lines. The complex 2 was also studied by NMR for its solution stability and by ESI-MS experiments for its ability to interact with biomolecules, such as cysteine, glutathione or guanosine 5'-monophosphate.The platinum(II) oxalato complexes [Pt(ox)( Platinum carboxylates represent a notable group of transition metal complexes, which have been used for the treatment of various types of cancer for many years ,2,3. Vari.e., N-donor ligands) and leaving  ligands is one of the crucial challenge of modern bioinorganic chemistry ; L1=2-chloro-N6-(2-methoxybenzyl)-9-isopropyladenine) and IC50(MCF7) = 3.6 µM ([Pt(ox)(L2)]; L2=2-chloro-N6--9-isopropyladenine). Utku et al. described the platinum(II) oxalato complexes with 2-phenylbenzimidazole ligand and their antibacterial and antifungal activity [et al. dealt with the [Pt(ox)(L3)]  and their cytotoxic effect against A549, B16-F1, B16-F10 and MDA-MB-231 cancer cells and BHK-21 and CHO non-cancer cells. The obtained results proved four reported oxalato complexes as less active against the named cancer cells (IC50(A549) = 12.6–60.6 µM, IC50(B16-F1) = 14.5–21.9 µM, IC50(B16-F10) = 16.6–38.0 µM, IC50(MDA-MB-231) = 16.6–25.1 µM) as compared with cisplatin (IC50(A549) = 2.7 µM, IC50(B16-F1) = 3.5 µM, IC50(B16-F10) = 4.2 µM, IC50(MDA-MB-231) = 1.4 µM)  (4Claza)2]+H}+ for 1), 678.9 m/z (3Braza)2]+H}+ for 2) and 679.0 m/z (4Braza)2]+H}+for 3), their adducts with different cations, namely 627.1 m/z (4Claza)2]+K}+ for 1), 611.1 m/z (4Claza)2]+Na}+ for 1), 716.9 m/z (3Braza)2]+K}+ for 2), 701.0 m/z (3Braza)2]+Na}+ for 2), 716.9 m/z (4Braza)2]+K}+ for 3) and 701.1 m/z (4Braza)2]+Na}+ for 3). The peak of the released N-donor ligand was found at 153.1 m/z , 197.0 m/z  and 197.1 m/z . The pseudomolecular peaks of the {[Pt(ox)(naza)2]−H}– species were detected by means of ESI− (electrospray ionization in the negative mode) mass spectrometry at 585.9 m/z (4Claza)2]−H}− for 1) and 676.0 m/z (3Braza)2]−H}– for 2, and: calc. 675.9; 85%; {[Pt(ox)(4Braza)2]–H}− for 3). The ESI- mass spectra of the studied compounds also contain the peaks whose mass correspond to the {[Pt(ox)(naza)]−H}− fragment (4Claza)]−H}− for 1; 478.1 m/z, calc. 477.9; 30%; {[Pt(ox)(3Braza)]−H}− for 2; 478.1 m/z, calc. 477.9; 65%; {[Pt(ox)(4Braza)]−H}− for 3) and {naza−H}− ligand .The platinum(II) oxalato complexes [Pt(ox)2] 1–3;  were pre1–3, as well as the starting compounds naza and K2[Pt(ox)2]∙2H2O, were studied by means of multinuclear and 2D NMR spectroscopy. All the 1H, 13C and 15N signals of free naza molecules were unambiguously detected in the spectra of corresponding platinum(II) complexes and assigned by means of the below-mentioned 2D NMR experiments. The significantly different 15N-NMR coordination shift values of the N1 (|Δδ| = 2.5–3.4 ppm) and N7 (|Δδ| = 114.0–115.1 ppm) atoms clearly proved the coordination of naza molecules through the N7 atoms. The 13C-NMR chemical shifts of the C11 and C12 atoms of the oxalate dianion (detected at 165.9–166.0 ppm) and the 195Pt chemical shifts, which equal −1770.1 (1), −1783.5 (2) and −1772.5 (3), correlate well with the values of the formerly reported platinum(II) oxalato complexes with 7-azaindole 2+ and/or cis-[Pt(OH)2(3Braza)2] in our case +H}+ species , which most probably contains a ring-opened product of the interaction with a monodentate bound oxalate dianion.ESI-MS was also used to study the ability of  mixture :1 v/v  complexes is based on the covalent binding of activated platinum(II) species to the nuclear DNA molecule of the tumour cells , which i1–3 were screened for their in vitro antitumor activity against two types of the human cancer cell lines - osteosarcoma (HOS) and breast adenocarcinoma (MCF7)  in the medium used. This compound showed the moderate in vitro anticancer activity, concretely 27.5 ± 3.4 μM against HOS and 18.3 ± 3.6 μM against MCF7 cells, which is comparable effect with that of cisplatin (IC50(HOS) = 25.4 ± 8.5 μM, IC50(MCF7) = 18.1 ± 5.1 μM)  > 50.0 μM, IC50(MCF7) > 50.0 μM). Nevertheless, it can be stated that the complex 2 exceeded the in vitro antitumor activity of oxaliplatin on HOS and MCF7 human cancer cell lines.The complexes a (MCF7) . In the  5.1 μM) . It has 2 was tested against next six human cancer cell lines, namely malignant melanoma (G361), cervix carcinoma (HeLa), ovarian carcinoma (A2780), cisplatin-resistant ovarian carcinoma (A2780R), lung carcinoma (A549) and prostate adenocarcinoma (LNCaP). It has been observed that its in vitro antitumor activity equals IC50 = 17.3 ± 3.1 μM , IC50 = 31.8 ± 6.2 μM , IC50 = 19.2 ± 3.7 μM  and IC50 > 50.0 μM  cell lines , 3-iodo-7-azaindole (3Iaza) and 5-bromo-7-azaindole (5Braza), which, similarly to 1 and 3, did not show any effect against both HOS and MCF7 cell lines up to the concentration of 10.0, 25.0, and 0.5 μM, respectively. Concerning all six platinum(II) oxalato complexes with different 7-azaindole derivatives together, it can be said that the biological activity, in terms of bioavailability, is strongly affected by the position of halogeno-substituent of the 7-azaindole moiety, because the solubility of the complexes with 3Claza, 3Braza and 3Iaza (10.0–50.0 μM) is significantly higher than that of the complexes involving the 7-azaindole derivatives substituted in the position 4 (4Claza and 4Braza) or 5 (5Braza), whose solubility did not exceed 1.0 μM in the medium used.As it is mentioned above, reported  analogou2[PtCl4]), potassium oxalate monohydrate (K2(ox)∙H2O), 4-chloro-7-azaindole (4Claza), 3-bromo-7-azaindole (3Braza), 4-bromo-7-azaindole (4Braza), cisplatin, oxaliplatin, cysteine (cys), reduced glutathione (GSH), guanosine 5'-monophosphate disodium salt (GMP) and solvents were purchased from Sigma-Aldrich Co.  and Acros Organics Co.  and used as received.Potassium tetrachloridoplatinate(II) .Electrospray ionization mass spectrometry (ESI-MS): Mass spectra were obtained on fresh methanol solutions and after 2 h and 12 h by an LCQ Fleet ion trap mass spectrometer using both the positive (ESI+) negative (ESI−) mode electrospray ionization technique . The 10 µM  solution of 2 in methanol was mixed together with the same volume of water , water solutions of the mixture of GSH (6 µM) and cys (260 µM) or water solution of GMP. 20 µL of the mixtures was analysed by means of flow injection analysis/mass spectrometry (FIA/ESI-MS) in both the positive and negative ionization modes 0 h, 2 h and 12 h after the preparation.NMR spectroscopy: 1H, 13C and 195Pt NMR spectra and two dimensional correlation 1H-1H gs-COSY, 1H-13C gs-HMQC, 1H-13C gs-HMBC and 1H-15N gs-HMBC experiments (DMF-7d solutions) were performed at 300 K on a Varian 400 device  at 400.00 MHz (1H), 100.58 MHz (13C), 86.00 MHz (195Pt) and 40.53 MHz (15N); gs = gradient selected, COSY = correlation spectroscopy, HMQC = heteronuclear multiple quantum coherence, HMBC = heteronuclear multiple bond coherence. 1H and 13C spectra were adjusted against SiMe4, while 195Pt spectra were calibrated against K2[PtCl6] in D2O found at 0 ppm. 1H-15N gs-HMBC experiments were obtained at natural abundance and calibrated against the residual signals of DMF . The splitting of proton resonances in the reported 1H-NMR spectra is defined as s = singlet, d = doublet, t = triplet, br = broad band, m = multiplet. The coordination shift  is calculated as Δδ = δcomplex − δligand. The stability studies of the DMF-7d and DMF-7d/H2O (9:1 v/v) solutions of 2 was carried out by means of 1H and 195Pt NMR after 1, 2, 3, 4 and 5 days of standing at laboratory temperature. 4Claza (for 1), 3Braza (for 2) or 4Braza (for 3) in 10 mL of hot (50 °C) ethanol was slowly poured into the solution of K2[Pt(ox)2]∙2H2O (0.5 mmol) in 10 mL of hot (50 °C) distilled water. The reaction mixtures were stirred at 50 °C for two days. The products, which formed, were filtered off, washed (5 mL of distilled water and 5 mL of ethanol) and dried at 40 °C platinum(II)Bis(4-chloro-7-azaindole)-κN7} Light grey solid; yield 80%; 1H-NMR : δ/Δδ = 13.41/1.34 , 8.71/0.49 , 7.98/0.30 , 7.37/0.17 , 6.79/0.21  ppm. 13C-NMR : δ/Δδ = 165.9 , 148.2/−1.6 (C-7a), 146.6/3.0 (CH-6), 138.4/3.8 (C-4), 129.3/1.9 (CH-2), 122.2/3.1 (C-3a), 117.5/1.9 (CH-5), 100.5/2.2 (CH-3) ppm. 15N-NMR : δ/Δδ = 145.6/3.4 (NH-1), 154.6/−114.6 (N-7) ppm. 195Pt NMR : δ = −1770.1 ppm. ESI MS (30 ev): m/z = 611.1 (M+Na), 585.9 (M−H), 434.0 (M−4Claza−H), 153.1 (4Claza+H), 151.0 (4Claza−H). Anal. Calc.: C, 32.7%; H, 1.7%; N, 9.5%. Found: C, 32.8%; H, 1.6%; N, 9.6%.2O,O’)platinum(II)Bis(3-bromo-7-azaindole)-κN7} Light grey solid; yield 75%; 1H-NMR : δ/Δδ = 13.49/1.32 , 8.82/0.48 , 8.11/0.23 , 8.09/0.31 , 7.31/0.10  ppm. 13C-NMR : δ/Δδ = 166.0 , 147.5/3.2 (CH-6), 147.0/−0.9 (C-7a), 130.6/4.0 (CH-4), 128.0/2.1 (CH-2), 122.6/3.3 (C-3a), 118.0/1.4 (CH-5), 89.3/1.6 (C-3) ppm. 15N-NMR : δ/Δδ = 143.5/2.5 (NH-1), 159.4/−115.1 (N-7). 195Pt NMR : δ = −1783.5 ppm. ESI MS (30 ev): m/z = 678.9 (M+H), 676.0 (M−H), 478.1 (M−3Braza−H), 197.1 (3Braza+H), 195.1 (3Braza–H). Anal. Calc.: C, 28.4%; H, 1.5%; N, 8.3%. Found: C, 28.4%; H, 1.5%; N, 8.3%.2O,O’)platinum(II)Bis(4-bromo-7-azaindole)-κN7} Light grey solid; yield 80%; 1H-NMR : δ/Δδ = 13.40/1.31 , 8.60/0.47 , 8.00/0.30 , 7.51/0.16 , 6.72/0.21  ppm. 13C-NMR : δ/Δδ = 165.9 , 147.3/−1.6 (C-7a), 146.3/2.9 (CH-6), 129.4/1.9 (CH-2), 128.0/4.1 (C-3a), 124.6/3.2 (C-4), 120.6/1.8 (CH-5), 102.1/2.2 (CH-3) ppm. 15N-NMR : δ/Δδ = 145.5/3.1 (NH-1), 155.4/−114.0 (N-7). 195Pt NMR : δ = −1772.5 ppm. ESI MS (30 ev): m/z = 679.0 (M+H), 676.0 (M−H), 478.1 (M−4Braza−H), 197.1 (4Braza+H), 195.1 (4Braza−H). Anal. Calc.: C, 28.4%; H, 1.5%; N, 8.3%. Found: C, 28.3%; H, 1.5%; N, 8.4%.Breast adenocarcinoma , osteosarcoma , malignant melanoma , cervix epitheloid carcinoma , A2780 ovarian carcinoma (ECACC No. 93112519), A2780R cisplatin-resistant ovarian carcinoma (ECACC No.93112517), lung carcinoma  and prostate adenocarcinoma  cancer cell lines were purchased from European Collection of Cell Cultures .In vitro cytotoxicity was determined by an MTT assay against MCF7 (the complexes 1–3), HOS (1–3), G361 (2), HeLa (2), A2780 (2), A2780R (2), A549 (2) and LNCaP (2) human cancer cell lines. The cells were maintained in a humidified incubator . The cells were treated with 1–3 or standards  at the 0.01, 0.1, 1.0, 5.0, 25.0 and 50 μM concentrations for 24 h, using multi-well culture plates of 96 wells. In parallel, the cells were treated with vehicle  and Triton X-100  to assess the minimal  and maximal  cell damage, respectively. The MTT assay was measured spectrophotometrically at 540 nm . The data were expressed as the percentage of viability, when 100% and 0% represent the treatments with DMF and Triton X-100, respectively. The cytotoxicity data from the cancer cell lines were acquired from three independent experiments (conducted in triplicate) using cells from different passages. The IC50 values (µM) were calculated from viability curves. The results are presented as arithmetic mean ± SD.p < 0.05 considered to be significant .The significance of the differences between the results was assessed by the ANOVA analysis, followed by Tukey’s post-hoc test for multiple comparisons, with naza)2] (1–3) and evaluates their in vitro cytotoxicity on the selected human cancer cell lines. The testing revealed the complex 2 (involving 3Braza) as in vitro antitumor active against HOS, MCF7, G361, HeLa and A2780 with IC50° 17–32 µM. Because the complex 2 differs from 1 (involving 3Claza) and 3 (involving 4Braza) in the nature or position of the 7-azaindole moiety substituent, it can be concluded that 3Braza represents a very perspective N-donor ligand, which could be used as a carrier ligand involved into platinum(II) complexes with different leaving group than oxalato one.This work describes three new platinum(II) oxalato complexes [Pt(ox)(


Squamous cell carcinomas (SCCs) account for the majority of cancer mortalities. Although TP63 is an established lineage-survival oncogene in SCCs, therapeutic strategies have not been developed to target TP63 or it’s downstream effectors. In this study we demonstrate that TP63 directly regulates NRG1 expression in human SCC cell lines and that NRG1 is a critical component of the TP63 transcriptional program. Notably, we show that squamous tumors are dependent NRG1 signaling in vivo, in both genetically engineered mouse models and human xenograft models, and demonstrate that inhibition of NRG1 induces keratinization and terminal squamous differentiation of tumor cells, blocking proliferation and inhibiting tumor growth. Together, our findings identify a lineage-specific function of NRG1 in SCCs of diverse anatomic origin. Within the past decade, lineage addiction has emerged as a common paradigm to explain how certain tumors depend on co-opted survival and self-renewal programs that drive the normal development of the tissues from which they arise . During TP63 is prevalent in SCCs, and TP63 expression is used to distinguish SCCs from other cancer subtypes in multiple tissues  share many common properties, including genetic and epigenetic alterations . The TP6 tissues . SCCs ar tissues . DespiteHere we show that NRG1 expression is directly regulated by TP63 in SCCs of various organs, and that co-expression of NRG1 and its receptor ERBB3 is prevalent in SCCs. Moreover, we find that many of the SCC models that co-express NRG1 and ERBB3 depend on NRG1 autocrine signaling in vivo, in contrast to non-squamous cancers that exhibit NRG1 autocrine signaling but are not dependent on it.NRG1 and TP63 expression significantly correlated in both esophageal and lung squamous cell carcinomas (LUSC) as determined from The Cancer Genome Atlas (TCGA) transcriptome data (TP63 isoforms (siTP63 # 14) or only the TA-TP63 isoforms (siTA-TP63 # 13) and assessing expression of both isoforms of the NRG1 EGF-like domain, NRG1α and NRG1β, by qPCR in the OE-21 and KYSE-140 SCC cell lines. Knockdown of just the TA isoforms modestly reduced NRG1 expression, whereas silencing of all isoforms robustly decreased NRG1 expression , which encompasses the TP63 binding motif, compared to the control locus at −21 kB  compared to control treatment, nearly doubling time to progression from 14 to 27 days (p<0.002)  . Progresp<0.002) .NRG1/ERBB3 co-expression in ovarian cancer. We tested the effect of anti-NRG1 on a panel of ovarian cancer cell lines and indeed proliferation of cell lines expressing both NRG1 and ERBB3 was inhibited by anti-NRG1 in vitro  as described . Cell liAll animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at Genentech . For cell line xenograft studies, tumor cells were subcutaneously inoculated into C.B-17 SCID beige mice (FaDu) or athymic nude mice (HCC95 and KYSE-180). Mice were randomized into treatment groups when tumor volumes reached ~200mm3. Antibodies were dosed once per week intraperitoneally (i.p.) at 20 mg/kg for three doses, and tumor volumes and body weights were measured twice a week. For RNAseq, histology and western blot, HCC95 tumors were collected 72 hr after the first or third dose. For patient-derived tumor xenografts, tumor fragments were subcutaneously implanted into Balb/C nude mice, which were randomized into treatment groups (n = 5) when tumor volumes reached 150–250 mm3. Dosing and measurements were performed as noted above for 3–4 weeks.CreERT2+; tdTomato; KrasG12Dwt/ki; PTENloxP/loxP squamous skin cancer mouse model (manuscript in preparation), was used following Genentech IACUC guidelines. At 10–15 weeks of age, mice were dosed once with Tamoxifen . Three days after a single tamoxifen dose, mice were divided into two groups with mice of similar proportion of age and sex, and dosed with anti-Ragweed or anti-NRG1 antibodies .The Lgr53). Percent tumor growth inhibition (TGI) was calculated relative to the average tumor volume of control (anti-Ragweed) mice. Kaplan-Meier analysis was used to determine progression-free survival for the skin GEMM SCC mice. Log-rank analysis was used for statistical analysis to compare treatment groups.A mixed-modeling approach was used to analyze tumor volumes for xenograft and PDX studies as explained earlier . Cubic rGGACAGCAGCAUUGAUCAAUU, AS = UUGAUCAAUGCUGCUGUCCUU), #2 ], and negative control non target control  were purchased from Dharmacon. RNA was isolated after 72 hr using a RNeasy Plus kit (Qiagen). cDNA was prepared using the Advantage RT for PCR kit (Clontech), and qPCR was performed using ABI master mix, and the results were validated by three independent experiments using the following Taqman assays (Life Technologies): TP63-all (Hs00978340_m1), TA-TP63-TA (Hs00186613_m1), deltaN-TP63 (Hs00978339_m1), NRG1a (Hs01103794_m1) and NRG1b (Hs00247624_m1).Cells were plated in medium without antibiotics and transfected with 5 nmol/L of siRNA using Dharmafect transfection reagent (Dharmacon). siRNAs for TA-TP63 (#13 = J-003330–13), all isoform-TP63 (#14 = J-003330–14), deltaN-TP63 [#1 . Proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane and stained with the indicated antibodies. HCC95 tumors from the in vivo studies were processed similarly for analysis of pharmacodynamic and apoptotic markers. Antibodies: TP63 alpha , deltaNTP63 (Biolegend 619001), actin (BD Bioscience 612656), p-ERBB3 (CST 4791), ERBB3 (CST 12708), PARP (CST 9542) and Cleaved caspase-3 (CST 9664).5’-TTCAAAAGGGAGTGCCAACTTTTCC-3’, 5’-GGTGCCTCACCTTTCTTCTTCCTGTCC-3’) and −30 KB NRG1 promoter  from NRG1 transcript start site as described earlier . Cultures of 25 million cells were fixed and cross-linked with formaldehyde (1.1%) for 15 min at room temperature and stopped with glycine. The cell pellet was re-suspended in ChIP lysis buffers and sonicated using a Bioruptor sonicator (Diagenode) to produce chromatin fragments averaging 200–500 bp. Sheared chromatin was incubated overnight at 4°C with protein A-coated magnetic beads plus either anti-TP63 alpha (CST 13109), anti-deltaNTP63 (BioLegend 619001) or isotype control antibody. Beads were washed, DNA was eluted and crosslinks were reversed during an incubation overnight at 65°C. Samples were treated with iPure beads and DNA was purified. Quantitative PCR was performed using sybr green PCR master mix (Applied Biosystem) to assess enrichment at the NRG1 promoter. The results were validated by three independent experiments. The ChIP ratio was calculated as enrichment over noise, normalized to the input. Statistical significance was determined by one-way ANOVA. Primers used for CHIP PCR were −21 kB NRG1 promoter  antibody , incubated at a concentration of 1.0 ug/mL for 60 min at room temperature and binding was visualized using ABC-Peroxidase Elite followed by DAB chromagen and counter stained with Mayer’s hematoxylin. KRT10 expression was reviewed manually by a translational pathologist (JMG) and scored for the percentage of cells demonstrating moderate to strong immunoreactivity, excluding areas of necrosis (0–100%).RNA-seq libraries were prepared using TruSeq RNA Sample Preparation kit  and sequenced on Illumina HiSeq 2500 sequencers, yielding an average of 34 million single-end reads (50 bp) per sample. Reads were aligned to the human genome version NCBI GRCh37 using GSNAP. Expression counts per gene were obtained by counting the number of reads aligned concordantly within a pair and uniquely to each gene locus as defined by NCBI, Ensembl gene annotations, and RefSeq mRNA sequences. Differential gene expression analysis was performed using edgeR. Gene enrichment analysis was performed on the edgeR differential expression results using the Gsea Preranked tool available through the Broad’s GSEA application. DESeq was used to compute the variance stabilized expression values for plotting the expression heat maps.Graphical and statistical data were generated with Microsoft Excel or GraphPad Prism . Statistical significance of differences between the results was assessed using a standard 2-tailed t-test or one-way ANOVA using Prism. p<0.05 was considered statistically significant. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Kevin Struhl as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "NRG1 is a critical component of the TP63 transcriptional program driving squamous cell cancers in multiple tissues" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The reviewers believe that the findings are of potential significance and may have therapeutic implications. Although NRG1 has already been established as a TP63 target in breast cells and NRG1/ERBB signaling has already been reported to play a role in squamous cell carcinoma biology, a strength of the work is the use of human material, murine models and cancer cell lines as well as an antibody to block NRG, which suggests that blocking NRG may affect tumor maintenance. However, the reviewers request additional work to more clearly demonstrate the interaction of NRG and p63, to clarify the specificity of some of the reagents and to determine whether squamous differentiation is the basis of the observed findings.Essential revisions:1) The title is quite broad. The reviewers were unclear whether the findings are true in all squamous tissues or are specific for a particular squamous epithelium. This should be addressed, and the title should be changed to more accurately reflect the findings presented in the manuscript.2) Further evidence to support co-expression/co-localization of NRG and p63 in SCC would strengthen the manuscript.3) The use of the blocking antibody is powerful but the details of how specific this reagent is for NRG are lacking. Further evidence of specificity and/or the use of genetics to support the findings are necessary.4) The authors should clarify whether tumors regress or arrest after treatment with the antibody. A waterfall plot of all of the tumors would be helpful.5) Is there squamous differentiation in cell lines? This needs to be assessed. If so, it is surprising that this did not affect proliferation.6) Although several experimental models are used, it would be helpful to perform similar experiments in at least three cell lines or to determine whether there are specific squamous tissues in which these findings are most relevant.For your information, we have included the reviews below, as you might find them helpful when preparing the revised version of your manuscript.Reviewer #1:In this manuscript Jackson and co-workers investigated the role of p63-NRG in tumor cell proliferation and tumor maintenance. They found that p63 and NRG expression co-occurred in the TCGA lung and esophageal cancer datasets. They showed that suppressing p63 decreased NRG expression and was localized to the NRG. Using a NRG antibody they showed that there was a modest effect on cell proliferation but a stronger effect in some tumor models. They argue that the effects on tumors is due to SCC differentiation.Overall these experiments are well described and well controlled. The observations are timely and important and may have therapeutic implications. However, there are a few points that require some additional clarification.The authors argue that the NRG plays a key role in SCC or those that co-express NRG and p63. However, they do not show this directly. It would be helpful to perform experiments in a panel of cell lines in which these markers are noted so that the point can be made clear. This is particularly important since the authors show that some tumors don't respond to anti-NRG, suggesting that they don't co-express p63 and NRG but this is not shown.It is also not clear whether the tumors regress or simply arrest. It would be helpful to show a waterfall plot for each tumor.It is also not clear why squamous differentiation would not affect proliferation. Have the authors formally assessed whether there is evidence of squamous differentiation in the 2D cultures?Reviewer #2:The authors show that NRG1 is a transcriptional target of p63 and argue that anti-NRG1 could be an effective therapy for a wide variety of squamous cell carcinomas. This data is interesting; however, the authors use a very limited number of cell lines and in vivo models. To strengthen the manuscript, the authors should focus on one type of SCC with a more robust number of cell lines and in vivo models.Reviewer #3:In this manuscript Hedge et al. show that TP63 directly regulates NRG1 expression in squamous cell carcinoma (SCC) cell lines. In addition, they show that treatment with an anti-NRG1 antibody inhibits the growth of SCC xenograft models and provide evidence that inhibition of NRG1 induces keratinization and terminal squamous differentiation of tumor cells. Although the data are potentially interesting, there are some major issues with the experimental design, which include the following:1) All experiments aimed at elucidating NRG1 function utilize an NRG1 blocking antibody. However, the authors fail to demonstrate (e.g. by using genetic tools) that the phenotype observed in their model systems upon antibody treatment is specifically mediated by NRG1 inhibition.2) NRG1 has already been established as a TP63 target in breast cells and NRG1/ERBB signaling has already been reported to play a role in squamous cell carcinoma biology. The authors provide some evidence that NRG1 inhibition decreases SCC cell proliferation and promotes cell differentiation. This hypothesis should be further investigated in properly controlled in 3D cell culture models where the role of ERBB3 and ERBB4 signaling could also be addressed.3) In Figure 1, it would be important to perform double IHC/IF to demonstrate co-localization of deltaN-TP63 and NRG1 at the protein level in tumor cells within human SCC tissue samples.Nrg1/ErbB signaling in the treated in vivo models should be provided.4) In Figure 3, immunoblotting data documenting inhibition of 5) In Figure 4, data for anti-Ragweed control (3 dose) should be showed for all experiments. Essential revisions:1) The title is quite broad. The reviewers were unclear whether the findings are true in all squamous tissues or are specific for a particular squamous epithelium. This should be addressed, and the title should be changed to more accurately reflect the findings presented in the manuscript.We revised the title to better reflect the findings. The new title is “NRG1 is a Critical Regulator of Differentiation in TP63-driven Squamous Cell Carcinoma”.2) Further evidence to support co-expression/co-localization of NRG and p63 in SCC would strengthen the manuscript.This is a great suggestion to demonstrate the co-localization of NRG1/p63. Unfortunately, the available NRG1 antibodies do not work for immunofluorescence, IHC or even western blot. We have tested many different antibodies for NRG1 in the past and have found them to be non-selective. We always perform shRNA knockdown of NRG1 to validate any commercial antibodies and so far, we have not found one that shows a specific staining pattern. Moreover, the blocking antibodies used in this paper are also not suitable for those applications. However, we would like to bring to the reviewer’s attention that we have provided TP63, NRG1, ERBB3 and ERBB4 mRNA levels in a table format just below the in vitro growth assay in Figure 2.3) The use of the blocking antibody is powerful but the details of how specific this reagent is for NRG are lacking. Further evidence of specificity and/or the use of genetics to support the findings are necessary.Details on antibody selectivity can be found in our earlier publication . We have added a sentence pointing the readers to this publication for additional details on the antibody properties.4) The authors should clarify whether tumors regress or arrest after treatment with the antibody. A waterfall plot of all of the tumors would be helpful.We have included a waterfall plot of all tumors as a supplementary figure. The tumors indeed undergo stasis not regression, consistent with the role of NRG1 in regulating differentiation, not cell survival. As discussed in our final paragraph, EGFR and other signaling pathways also play an important role in driving these tumors and inhibition of NRG1 could drive deeper responses when combined with EGFR inhibition.5) Is there squamous differentiation in cell lines? This needs to be assessed. If so, it is surprising that this did not affect proliferation.We thank the reviewers for raising this critical question. We carried out qPCR analysis of four differentiation markers in three different squamous cell carcinoma lines treated with anti-NRG1 or control antibody in vitro. The result are provided in Figure 4—figure supplement 3. Consistent with our in vivo findings, the cell lines treated in vitro also showed significant increases in multiple differentiation markers. However, the magnitude of induction was much lower in vitro, consistent with the more modest effects on proliferation observed in vitro compared to the tumor growth inhibition in vivo.6) Although several experimental models are used, it would be helpful to perform similar experiments in at least three cell lines or to determine whether there are specific squamous tissues in which these findings are most relevant.We have conducted additional experiments as mentioned above with 3 cell lines; FaDu (Head and Neck), HCC95 (lung) and KYSE-180  squamous cell carcinomas. The induction of differentiation markers upon treatment was highest in the lung and esophageal lines.


NAC1 is a cancer-related transcription regulator protein that is overexpressed in various carcinomas, including ovarian, cervical, breast, and pancreatic carcinomas. NAC1 knock-down was previously shown to result in the apoptosis of ovarian cancer cell lines and to rescue their sensitivity to chemotherapy, suggesting that NAC1 may be a potential therapeutic target, but protein complex formation of intranuclear NAC1 in ovarian cancer cells remain poorly understood. In this study, analysis of ovarian cancer cell lysates by fast protein liquid chromatography on a sizing column showed that the NAC1 peak corresponded to an apparent molecular mass of 300–500 kDa, which is larger than the estimated molecular mass (58 kDa) of the protein. Liquid chromatography-tandem mass spectrometry analysis identified CARM1 as interacting with NAC1 in the protein complex. Furthermore, tissue microarray analysis revealed a significant correlation between CARM1 and NAC1 expression levels. Ovarian cancer patients expressing high levels of NAC1 and CARM1 exhibited poor prognosis after adjuvant chemotherapy. Collectively, our results demonstrate that high expression levels of NAC1 and its novel binding partner CARM1 may serve as an informative prognostic biomarker for predicting resistance to chemotherapy for ovarian cancer. Ovarian cancer is the most aggressive gynecological malignancy worldwide  and its NACC1 gene, is a nuclear protein that encompasses an N-terminal BTB/POZ  (hereafter abbreviated BTB) and a C-terminal BEN  domain. The BTB domain is a ∼100 amino acid highly conserved motif that mediates homodimerization and/or heterodimerization and interacts with other proteins SAM as a methyl donor. The analysis clearly showed that CARM1 methylates histone H3, but not GST or GST-NAC1  enzyme that is known to methylate histone H3 (H3R17me2a and H3R26me2a), and nonhistone substrates . We ther1 Figure . To deteP < 0.0001. The significant correlation of CARM1 and NAC1 expression levels implies that NAC1 functions synergistically with at least CARM1 to promote tumorigenesis in ovarian cancers.In an attempt to determine the expression levels of CARM1 and NAC1 in clinical specimens, we performed immunohistochemistry (IHC) for CARM1 and NAC1 in 84 ovarian carcinoma tissues. Both antibodies stained tumors more strongly than the surrounding benign tissues Figure . High exP = 0.0152 and P = 0.0091, respectively) , residual tumor ≥1 cm , and high NAC1/CARM1 expression  correlated with shorter progression-free and overall survival (Tables P = 0.0002) for shorter progression-free survival  for overall survival  Figure . UnivariThe knockdown of CARM1 as well as NAC1 using siRNA suppressed cell growth in the ovarian carcinoma cell lines A2780 and MDAH2774 Figure .The present study indicates that NAC1 forms 300–500 kDa protein complexes in ovarian cancer cells, comparable with that of HeLa cells . The moset al. reported that CARM1 overexpression was noted only in a small number (17%) of ovarian cancer patients -adenosyl-L-methionine  (for hot assay) or 20 µM S-adenosylmethionine (SAM) (for cold assay) in methylase activity buffer  was incubated for 60 min at 30° C. The reaction products were separated by 15% SDS-polyacrylamide gel electrophoresis, followed by Coomassie Brilliant Blue staining and autoradiography (for hot assay) or by Western blotting with anti-histone H3R17me2a (for cold assay).Reaction mixture (30 µl) containing 1 µg of different recombinant human histone H3, GST and GST-NAC1 (as substrates), the indicated immunoprecipitate (as enzyme), and 1 µL of 2 or docetaxel 70 mg/m2). All patients received 6-12 courses of this combination regimen. The Shimane University Institutional Review Board approved the acquisition of tumor tissues and written informed consent was obtained from all subjects.Formalin-fixed, paraffin-embedded tissue samples of 84 ovarian cancers, including 44 serous carcinomas, 10 mucinous carcinomas, 10 clear cell carcinomas, and 20 endometrioid carcinomas, were used in the present study. Diagnoses were based on conventional morphological examinations of sections stained with hematoxylin and eosin (H&E), and tumors were classified according to the WHO classification. Tumor staging was performed according to the International Federation of Gynecology and Obstetrics (FIGO) classification. Samples were obtained from the Department of Obstetrics and Gynecology at Shimane University Hospital. All patients were primarily treated with cytoreductive surgery and adjuvant platinum and taxane chemotherapy  and pathology technician (K. I.) and was based on a review of H&E slides. The immunohistochemistry method and the evaluation criteria were detailed in a previous study .t-test was used to examine the significance of differences in growth assay data. Chi-squared test or Fischer’s exact test was used for comparisons of categorical data. Data are presented as means ± SD. P < 0.05 was considered significant.Progression-free and overall survivals were calculated from the date of diagnosis to the date of first relapse or last follow-up. Age and performance status distributions were similar between patients expressing and not expressing NAC1 or CARM1. Data were plotted as Kaplan–Meier curves, and the significance of differences was determined using the log-rank test. A multivariate prognostic analysis was performed using a Cox proportional hazards model. Data were censored when patients were lost to the follow-up. Student’s


Escherichia coli at different antibiotic concentrations for ∼400 generations. Our results reveal fast evolution toward specialization following the first mutational step toward resistance, along with pervasive trade‐offs among different evolution doses. We found clear and regular fitness patterns of specialization, which converged rapidly from different initial starting points. These findings are consistent with a simple fitness peak shift model as described by the classical evolutionary ecology theory of adaptation across environmental gradients. We also found that the fitness costs of resistance tend to be compensated through time at low doses whereas they increase through time at higher doses. This cost evolution follows a linear trend with the log‐dose of antibiotic along the gradient. These results suggest a general explanation for the variability of the fitness costs of resistance and their evolution. Overall, these findings call for more realistic models of resistance management incorporating dose‐specialization.Antibiotic and pesticide resistance of pathogens are major and pressing worldwide issues. Resistance evolution is often considered in simplified ecological contexts: treated versus nontreated environments. In contrast, antibiotic usually present important dose gradients: from ecosystems to hospitals to polluted soils, in treated patients across tissues. However, we do not know whether adaptation to low or high doses involves different phenotypic traits, and whether these traits trade‐off with each other. In this study, we investigated the occurrence of such fitness trade‐offs along a dose gradient by evolving experimentally resistant lines of  The massive increase in use of antibiotics since 1950 has greatly improved standards of living in our societies but has led to an explosion in the number of resistant phenotypes in microorganisms   for ∼400 generations in order to “push” them close to their optimal phenotype. We started with a susceptible reference strain, or “SRef” strain, corresponding to the 10,000th (abbreviated 10 K) generation of Lenski's long‐term adaptation experiment (E. coli strain REL4536). First, we obtained a highly resistant reference strain (“RRef” strain) by screening twice SRef for antibiotic resistance . We then evolved two series of strains at the different Nal doses. In the first main series, six resistant lines per ED  were initiated from independent resistant mutants of SRef obtained after a screen at the same dose than the ED . These lines (hereafter “SRef lines”) provided a diversity of first mutational step and evolved from the screen at a given Nal dose. In the second series, two lines per ED  were initiated from the RRef strain. These lines (hereafter “RRef‐lines”) provided with replicated evolution of the same initial resistant mutant , corresponding to the 10,000th generation of Lenski's long‐term adaptation experiment to Davis minimal broth DM25 (Lenski and Travisano All lines were derived from the −7). They were obtained via an additional fluctuation assay  divided among the five evolution doses and the six control lines  evolving at dose 0. All lines were distributed into four 24‐wells lidded microplates with 1 mL medium per well, with the six Nal concentrations allocated in each plate. Inoculated wells were alternated with noninoculated wells following a checkered pattern, to control for contaminations. The plates were incubated at 37°C, 200 rpm in a water‐saturated atmosphere . Every 24 h, 10 μL of the cultured lines were transferred to new wells containing 1 mL of fresh medium and incubated in the same conditions. The protocol was applied for two months corresponding to ∼400 generations of evolution after what the lines were stored as glycerol stocks.gyrA gene at Tini and Tfin. This sequence covers most of the promoter and 32% of the gyrA gene and its complete quinolone resistance‐determining region . Sequencing was performed using Eurofins mwg operon . Mutations in gyrA gene were identified by comparing the mutant sequences to that of the SRef strain.All lines were sequenced at the ini and Tfin. The “fitness profile” of each line (YFP) across the dose gradient was determined by competitions against the RRef‐CFP strain in DM250 at the five measure doses . Ecotoxicological measures such as MIC (minimum inhibitory concentration) are often used to characterize resistance evolution. However, competitive fitness is better suited in our context, to quantify adaptation and the evolution of specialization. For instance, two lineages with similar MIC can have very different competitive abilities, below their common MIC. The measure dose 150 was preferred to dose 200 because the latter exhibited high growth inhibition of the RRef strain, such that fluorescence measurements were close to the detection limit. The fitness costs of resistance were measured from competitions in absence of Nal (dose 0) against the strain SRef‐CFP.Competitions experiments were performed from glycerol stocks at Tt0). Competitions were initiated by inoculating 2 μL of the competition mix into 200 μL of DM250 . Fluorescence signals were measured again after 24h of growth (t24) in the same conditions as the evolution experiment . Each competition was repeated at least four times at different dates. We assumed that fitness effects were transitive between genotypes competing in the same dose. Deviation from transitivity as measured in Gallet et al. (−3 at most in these experiments).Competition assays were performed in the following procedures. 1:1 volumic ratio competition mixes were prepared from saturated cultures. Fluorescence signals of these mixes were measured on a Tecan Infinite 200  prior to the competition .The selection coefficient per generation associated with each competition was estimated as:lineID, MD (six doses) and time (Tini and Tfin). Random effects included plate and date of the competition assay. The model was used to estimate the fitness values at Tini and Tfin, the fitness changes between Tini and Tfin (pairwise comparisons) of each line in each MD, and their standard errors (performed with mvt adjustments with the lsmeans R package), which are all represented in figures. Polynomial fits of the fitness profiles of evolved lines (from the fitness estimates at Tfin of the previous model) in each MD were performed as functions of the log‐ED of lines (nls function of the R Stats package). The fits were done for the SRef lines alone or for both the RRef and the SRef lines for the comparison of the final fitness profiles of lines evolved in the same ED but initiated from mutants screened at different doses.Fitness measures were analyzed in a linear‐mixed model  with fixed effects on P value = 0.773) nor between replicates   due to adaptation to experimental conditions . The last replicate differed only slightly from the three others due to a fitness jump at generation 400 . The global fitness trajectory of the control0K lines is consistent with that expected from the similar 400 generations evolution in DM25 in the LTEE  Fig. . This reE. coli, the DNA gyrase is the target of Nal and many of the known resistance mutations occur in the gyrA gene coding for the GyrA subunit of this enzyme  and some non‐gyrA mutations (four lines) were initially sampled across the sets of single resistant mutants screened at the doses 3, 8, and 20 .In  20 Fig. . Evoluti 20 Fig. . In all gyrA mutants 87Gly and 119Glu) went extinct early in the evolution experiment. One of the SRef‐lines evolving at ED20 proved to be initially polymorphic for the gyrA resistance mutation. This line was removed from later analysis in order to avoid confusion in the data interpretation. One of the two RRef‐lines evolving at Nal3 was contaminated at one point and removed from the experiment. In all other lines, no additional mutations than the ones detected at Tini were found in the gyrA sequence at Tfin. Among the four lines without gyrA mutations initially (hereafter “non‐gyrA” lines), only one line acquired a gyrA mutation (87Tyr) during the evolution experiment.Two SRef‐lines evolving at ED200 , all fitness profiles show a regular quadratic pattern with log‐ED. Fitted curves are shown on each panel and Table gyrA mutation show a large fitness lag compared to all other lines, at all MD. Given this large and consistent difference, these lines were not included in the fit of the average fitness profiles among ED represented on Figure gyrA mutations after the initial screen, despite that these mutations are frequent and provide a strong fitness benefit. The effect of the first mutational step is also visible on gyrA lines, but is very small. In several cases, lines with the same initial gyrA mutation tend to group together for a given ED and MD . Similarly, RRef lines , tend to group together as well. These effects remain, however, minor compared to the overall trend. Even the RRef lines, which had a quite different starting point than most lines, show a final pattern very consistent with the general trend of SRef lines  and generation 400 of evolution (Tfin) in the MD corresponding to its ED. SRef lines evolved at ED200 are not shown as their fitness were not measured at MD200 (see Methods). For all lines, the fitness change was positive or not significantly different from zero at the dose in which they evolved. Among gyrA lines, the trend shows striking regularity independently of the evolution dose and of the identity of the gyrA mutation carried initially , with a slope close to –1. The trend is also observed among non‐gyrA lines but with a different intercept  and similar slope of about –1. This pattern is expected when evolution corresponds to convergence to a fitness peak, as it indicates that the fitness changes entirely compensate initial maladaptation. This pattern excludes a strong role for “chance” or transient “historical” effects  and show an adaptation (or no change) in all MD, indicating that evolving at low dose increases fitness at high dose. A different pattern is observed for lines evolved at high doses. While they adapted to their ED, they consistently and regularly “deadapted” to low doses.Studying a large sample of mutants screened at different doses in the same system showed that little dose specialization occurred after a single mutational step  but higher for non‐gyrA mutations. They were also variable between single‐mutants and “double”‐mutants of ED100 and ED200, even when they carried the same gyrA mutation. The change in cost between Tini and Tfin is negatively correlated with the ED . The non‐gyrA lines (present only at ED3) have generally higher costs than the gyrA lines . Furthermore, phenotypes evolved at intermediate doses are better adapted to high doses than phenotypes evolved at low doses (consistent with a multiple hit effect). Additionally, long‐term selection at low doses will not result in high fitness  at high doses, and as a result, long‐term selection at high doses will not select for high‐fitness phenotypes at low doses. These conclusions have important implications for understanding and modeling the evolution of resistance in the field, under heterogeneous conditions.To our knowledge, no previous study has clearly established the pattern of dose‐specialization along a gradient so far. First, many studies have investigated the fitness of resistant mutants from field samples . After 400 generations of evolution at ED3, their fitness profiles are still largely different to those of the gyrA lines indicating a strong historical contingency between those “mutational modules” of resistance. Contrary to the specialization pattern, this divergence in fitness patterns between the gyrA and the non‐gyrA lines was already observed immediately after the first resistance screen and interpreted as the occurrence of selective covariances across traits in Harmand et al. gyrA and non‐gyrA resistant lines as good candidates to investigate the effects of historical contingency and selective covariances on long‐term adaptive trajectories.“Historical contingency” or “mutation‐order” effects have been widely discussed in the literature to describe the dependence of adaptive trajectories on initial conditions  often overlook the possibility that dose gradients may represent different phenotypic challenges, in addition to also representing different selection intensities. Our results show that this simplification is not warranted. They show that the evolution of resistance along antibiotic dose gradients is consistent with classic evolutionary models of adaptation on ecological gradients, where each environment corresponds to one fitness peak. The observed patterns of adaptation and maladaptation are also fully consistent with the occurrence of dose‐dependent optima and show pervasive trade‐offs across doses. Hence, our findings call for more realistic models of resistance evolution in heterogeneous dose conditions that include dose‐specialization. Such models are necessary to better evaluate the impact of low doses of antibiotics that are today ecologically widespread.The authors declare no conflict of interest.Associate Editor: K. LythgoeFig. S1. Selection coefficients of control lines evolved in the absence of antibiotic in competition against the non‐evolved wild‐type 10K‐YFP (or 10K‐CFP in the case of 0K‐YFP lines).Click here for additional data file.Fig. S2. Fitness change of the SRef lines during 400 generations of evolution in different evolution doses (colors) versus their fitness just after the screen of resistance in the different measure doses. Symbols indicate the gyrA mutations sequenced after evolution in each line while colors indicate the dose at which they were screened and evolved. Error bars represent standard errors of the mean and the fitness change estimated in the statistical model.Click here for additional data file.Figure S2bClick here for additional data file.Figure S2cClick here for additional data file.Figure S2dClick here for additional data file.Figure S2eClick here for additional data file.Fig. S3. Costs of resistance of the SRel lines evolved for 400 generations at five evolution doses of antibiotic. Symbols indicate the mutation detected in the gyrA sequence or the absence of mutation in the gyrA sequence while colors indicate the evolution dose. The dotted horizontal line at 0 corresponds to an equal fitness with the susceptible ancestor 10K, while negative values correspond to a lower fitness of resistant lines. Error bars represent standard errors among replicates.Click here for additional data file.


The data presented in this article are related to the research paper entitled “Increased expression of mitochondrial sodium-coupled ascorbic acid transporter-2 (mitSVCT2) as a central feature in breast cancer”, available in Free Radical Biology and Medicine Journal [1]. In this article, we examined the SVCT2 transporter expression in various breast cancer cell lines using RT-PCR and Western blot assays. In addition, we analyzed the subcellular localization of SVCT2 by immunofluorescence colocalization assays and cellular fractionation experiments. Finally, an analysis of different cancer tissue microarrays immunostained for SVCT2 and imaged by The Human Protein Atlas (https://www.proteinatlas.org) is presented. Analysis of SVCT2 expression by RT-PCR and Western blot in four breast cancer cell lines are shown . Immunof22.1l-glutamine, penicillin/streptomycin (100 U/ml) and fungizone.Breast adenocarcinoma cell lines ZR-75, MCF-7, MDA-231 and MDA-468 were grown in DMEM:F12 (1:1) medium. Colon carcinoma cell line CaCo-2 was grown in DMEM medium. Embryonic kidney cell line HEK-293 was grown in DMEM-high glucose medium. All media were supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 1% 2.2PCR experiments were performed using the protocol described in Ref. 2.3Immunoblotting experiments were performed using the protocol described in Ref. 2.4Immunofluorescence experiments were performed using the protocol described in Ref. 2.5Cellular fractionation and mitochondria purification were performed using the protocol described in Ref.


Crotalus durissus collilineatus venom was isolated, structurally characterized and evaluated for its toxicity and ability to interfere with cell proliferation and migration in MDA-MB-231, a human breast cancer cell line.In recent decades, snake venom disintegrins have received special attention due to their potential use in anticancer therapy. Disintegrins are small and cysteine-rich proteins present in snake venoms and can interact with specific integrins to inhibit their activities in cell-cell and cell-ECM interactions. These molecules, known to inhibit platelet aggregation, are also capable of interacting with certain cancer-related integrins, and may interfere in important processes involved in carcinogenesis. Therefore, disintegrin from Based on previous studies, disintegrin was isolated by FPLC, through two chromatographic steps, both on reversed phase C-18 columns. The isolated disintegrin was structurally characterized by Tris-Tricine-SDS-PAGE, mass spectrometry and N-terminal sequencing. For the functional assays, MTT and wound-healing assays were performed in order to investigate cytotoxicity and effect on cell migration in vitro, respectively.p < 0.05), compared to negative control.Disintegrin presented a molecular mass of 7287.4 Da and its amino acid sequence shared similarity with the disintegrin domain of P-II metalloproteases. Using functional assays, the disintegrin showed low cytotoxicity  after 24 h of incubation and in the wound-healing assay, the disintegrin (3 μg/mL) was able to significantly inhibit cell migration (24%, C. d. collilineatus induces low cytotoxicity and inhibits migration of human breast cancer cells. Therefore, it may be a very useful molecular tool for understanding ECM-cell interaction cancer-related mechanisms involved in an important integrin family that highlights molecular aspects of tumorigenesis. Also, non-RGD disintegrin has potential to serve as an agent in anticancer therapy or adjuvant component combined with other anticancer drugs.Thus, our results demonstrate that non-RGD disintegrin from  Trimeresurus gramineus venom, called “trigramin”, presenting the arginine-glycine-aspartic acid (RGD) domain, was able to inhibit platelet aggregation, by preventing the connection between fibrinogen and platelets stimulated by ADP -2, 5 diphenyl tetrazolium bromide (MTT) assay according to Mosmann . Initial5 cells/well) were grown to 90% confluence on a 12-well plate. Then, with the aid of a sterile tip, a cicatrix was performed on the adherent monolayer of tumor cells. The culture medium was changed to remove the cell debris and a new culture medium added containing the lowest concentration (3.0 μg/mL) of disintegrin which demonstrated effects on the cell line in the MTT assay and PBS as CN. Images of the wounds were recorded with a camera coupled to the inverted microscope at times 0, 4, 8 and 24 h of incubation. The mean distance of the internal area was determined as the percentage migration using the software AxionVision according to the following equation:For the wound healing assay, 3.0 μg/mL of disintegrin was used, and the assay was performed according to Liang, Park and Guan . MDA-MB-n = 3) were statistically analyzed using the software GraphPad Prism 5 . After assessing normality of variable distributions using the Kolmogorov–Smirnov test, experimental data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Statistical significance was considered at p < 0.05.All the results were expressed as the mean ± standard deviation (SD). Data from three independent experiments . The electrophoretic profile revealed that Fraction 2 exhibits a protein band of apparent 7 kDa molecular mass and some contaminants Fig. . After tThe first 43 amino acid residues of the N-terminal region of the Fraction 6 were sequenced by Edman degradation and shared identity with others disintegrins Fig. . In addiC. d. collilineatus (C0L2T8) and C. d. durissus (Q2QA03), including the conservation of cysteine amino acid residues  of metalloproteases PII from ues Fig. . Comparit test - data not shown), we chose to use the concentration of 3 μg/mL in the wound healing assay.Cell viability of MDA-MB-231 was determined in the presence of disintegrin; it was observed that the concentrations of 3 and 6 μg/mL of disintegrin significantly decreased cell viability in approximately 15% and 17%, respectively Fig. . Considep < 0.05 compared to negative control), after 24 h of incubation  [C. simus (7.1 kDa) [C. d. collilineatus [Non-RGD disintegrin from 7.1 kDa) , disinte7.1 kDa)  and protlineatus , 40.Cdc non-RGD disintegrin isolated in this study is absent from the classical RGD motif or any other motif previously observed in this protein family or glutamate-cysteine-aspartate (ECD) motif which characterize disintegrin-like proteins that may inhibit tumor progression [C. d. collilineatus by transcriptome techniques [C. d. collilineatus (C0L2T8) and C. atrox (Q2QA03). On the other hand, these disintegrins, as well as that of our study, present the disulfide bond pattern of this protein family, another signature that characterizes disintegrins. This pattern of disulfide bonds has already been well reported and is closely related to the formation of the protein loop and activity. If disintegrins are reduced and alkylated their biological activity is decreased [As to the primary sequence obtained through Edman degradation and mass spectrometry procedures, gression . In the chniques , as wellecreased –53.IIbβ3 platelet integrins, preventing their binding to fibrinogen and consequently inhibiting platelet aggregation [IIbβ3) and breast cancer cells (αvβ3) are similar, disintegrins are considered candidates to be anticancer agents [Disintegrins are known to be potent inhibitors of platelet aggregation, and some drugs based on its structure were developed and approved by the FDA , such asr agents . Integrir agents . They arr agents , and arer agents .2 and β5 and moderate levels of αv, α3 and β1 integrins [5 and αvβ5 integrins when compared with non-breast cancer cell line (Hek-293) [C. d. colilineatus venom presents low cytotoxicity, although its toxicity increases with the concentration, having a dose-response effect. As in our results, the tzabcanin also showed dose-dependent toxicity against human malignant melanoma (A-357) and human colorectal adenocarcinoma (Colo-205) cell lines, but the viabilities of human breast adenocarcinoma (MCF-7) and human lung adenocarcinoma (A-549) cell lines were not affected [Since disintegrins are considered candidates to be agents of anticancer therapy, we performed cytotoxicity and cell migration assays on metastatic breast cancer cells, namely MDA-MB-231 cells. This cell line is able to express low levels of the subunits αntegrins . TaheriaHek-293) . Our resaffected , 60. Lebaffected , 61.Cdc non-RGD disintegrin was able to inhibit the migration of MDA-MB-231 cells after 24 h of incubation, demonstrating that it is a possible anticancer agent with potential to inhibit the formation of breast cancer tumors. Similar results were obtained from tzabcanin, which inhibited cell migration of A-375 and A-549 cell lines [Crotalus viridis viridis, and r-mojastin 1, from Crotalus scutulatus scutulatus, that inhibited the migration of human pancreatic adenocarcinoma cancer cells (BXPC-3) [The cell migration assay showed that ll lines , r-Virid(BXPC-3) .In addition, considering the potential of disintegrins as anticancer therapeutic agents, they can also be used in combination with known chemotherapeutics. For example, echistatin combined with cisplatin (CDDP) was capable of increasing the expression of nuclear factor kappa B (NFκB), caspase-9 and caspase-3, leading to increased apoptosis in MDA-MB-231 cells. These data suggest that the combination of this disintegrin with CDDP may serve as a new type of anticancer therapy .C. d. collilineatus venom. The studies performed on the human breast cancer MDA-MB-231 cell line demonstrate that this disintegrin is able to inhibit cell migration, without severely affecting cell viability. Therefore, it may be a very useful molecular tool for understanding ECM-cell interaction cancer-related mechanisms involved important family of integrin that would elucidate molecular aspects of tumorigenesis. Also, this non-RGD disintegrin has potential to serve as an agent in anticancer therapy or adjuvant component combined with other anti-cancer drugs.In this study, we described the isolation and characterization of the first disintegrin with an LVN motif from


Silver nanoparticles (AgNPs) are widely used nanomaterials in both commercial and clinical biomedical applications, due to their antibacterial properties. AgNPs are also being explored for the treatment of cancer in particular in combination with ionizing radiation. In this work, we studied the effects of AgNPs and ionizing radiation on mitochondrial redox state and function in a panel of lung cell lines . The exposure to AgNPs caused cell cycle arrest and decreased cell proliferation in A549, BEAS-2B and Calu-1, but not in NCI-H358. The mitochondrial reactive oxygen species (ROS) and protein oxidation increased in a time- and dose-dependent manner in the more sensitive cell lines with the AgNP exposure, but not in NCI-H358. While ionizing radiation also induced changes in the mitochondrial redox profiles, in general, these were not synergistic with the effects of AgNPs with the exception of NCI-H358 and only at a higher dose of radiation.  Engineered nanoparticles, defined as materials having at least one dimension less than 100 nm in size, made of materials, including metals , are increasingly used for industrial application and as components of consumer products . BecauseAmong metallic nanoparticles, silver nanoparticles (AgNPs) are the most widely used for consumer, industrial, and biomedical applications because of their unique physical, chemical, optical, catalytic, antibacterial properties ,5,6. The2O2 to form highly reactive sulfenic acids [2-Coumarin (DCP-Net2C)) to specifically image sulfenylated proteins in mitochondria [Mitochondria are involved in the maintenance of cell viability and function and regulation of cell metabolism. Mitochondrial damage and dysfunction are implicated in aging ,29, and ic acids . As a fiic acids . Identifchondria , which eBecause the lung is a likely site for environmental and occupational exposure to AgNPs, in this work, we describe the effects of AgNPs and ionizing radiation on mitochondrial redox state and function in a diverse panel of lung cell lines. In order to understand and potentially mitigate long term adverse health effects that may occur as a result of exposure to AgNPs, especially following inhalation, it is important to examine the sublethal effects of AgNPs on mitochondria. As other ROS inducers, including ionizing radiation, can potentially increase the toxic effects of AgNPs ,39 we ha2, in Ham’s F-12K (Kaighn’s) medium  for A549, RPMI-1640 medium  for Calu-1 and NCI-H358 and DMEM medium  for BEAS-2B. Culture media for all cells was supplemented with 10% fetal bovine serum , 100 U/mL penicillin and 100 µg/mL streptomycin .The experiments were performed with a panel of four lung cell lines: Three cancer cell lines  and one transformed lung epithelial cell line (BEAS-2B). All cell lines were obtained from the American Type Culture Collection (ATCC). Cells were cultured at 37 °C under 5% COPrevious studies indicated that the size and coating of AgNPs could affect the toxicity profile of AgNPs in colorectal cancer and murine monocyte/macrophage cell lines . In cont137Cs (Cesium) irradiator  with indicated doses. The relative timing of AgNPs and IR treatment is described in each figure legend. The ionizing radiation (IR) was applied with a 444 TBq 12,000 Ci self-shielded 2O2) or 1 µM MitoSOX  and analyzed by flow cytometry. Changes in mitochondrial protein oxidation with AgNPs and IR exposure were similarly quantified by flow cytometry after treating the cells with 50 µM mitochondria-targeted probe DCP-Net2C [2. For analysis of mitochondrial ROS, the cells were treated with 10 µM MitoPY1  for 1 h then were washed thoroughly in PBS to remove AgNPs not bound or internalized by cells. Cells were fixed in 2.5% glutaraldehyde at 4 °C overnight. Next, fixed cells were scraped from the wells, pelleted, embedded in resin, cut into ultrathin sections (80 nm) and placed on copper coated formvar grids. All samples were imaged using an FEI Tecnai Spirit transmission electron microscope . Samples were imaged without additional staining to facilitate the detection of AgNPs.t-test was used to compare the mean fluorescence values between different conditions. After treatment, the cells were washed twice with Dulbecco’s phosphate-buffered saline (DPBS)  and detached from the plates with trypsin. For mitochondrial content and ROS analysis, the cells were resuspended live and kept on ice. For the analysis of mitochondrial protein oxidation and cell cycle, the cells were fixed for 5 min with cold (−20 °C) methanol or for 30 min with 70% ethanol, respectively. Fixed cells were washed with DPBS before resuspending for analysis. For the analysis of the cell cycle, cells were treated with 100 µg/mL RNAse  and stained with 50 µg/mL propidium iodide . The flow cytometry experiments were conducted with a BD FACS Canto II Cell Analyzer , and 10,000–50,000 cells were analyzed for each condition. Each experiment was repeated at least three times independently. Data were analyzed using the FCS Express 6 Flow software , and the Students’ t-test was used to compare the mean values between different conditions.The cell proliferation was analyzed with CyQUANT assay  according to the manufacturer’s protocol, and the fluorescence was read with a Spark microplate reader . The ATP content in cells was measured with CellTiter-Glo 3D assay  according to the manufacturer’s protocol, and the luminescence was read with the Spark microplate reader. All conditions were measured in three technical replicates, and each experiment was repeated independently three times. The Students’ 4 cells/well were plated on a Seahorse plate. The next day 10 µg/mL AgNPs were added to the assigned wells and incubated for 24 h. On the day of the experiment, the assay media, compounds , 1 μM for Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)  and 1 μM Antimycin A /Rotenone  and cells were prepared according to Seahorse protocols. The experiment was run with Seahorse XF24 , following injection order: (1) AgNPs or vehicle media (1 h reading); (2) Oligomycin; (3) FCCP; and (4) Antimycin A/Rotenone. After the Seahorse analysis, the cells were lysed with modified RIPA buffer  protease and phosphatase inhibitor tablets) and protein concentration was measured with bicinchoninic acid assay  for data normalization. Each experiment was repeated at least two times independently. The data were analyzed with Wave software , and the Students’ t-test was used to compare the mean fluorescence values between different conditions.The effects of AgNPs on the mitochondrial function were measured using the Seahorse Mito Stress Test following the manufacturer’s protocol. Briefly, the day before the AgNP treatment, 1.5 × 10p = 0.106 for AgNP with 2 Gy irradiation and p = 0.014 for AgNP with 5 Gy irradiation as compared with AgNP only).Changes in cell proliferation were measured using CyQuant proliferation-assay. The response to the exposure to AgNPs was both time- a and dosTo determine if the varied sensitivity of the four cell lines to AgNPs was due to differential uptake of AgNPs into cells, we examined the uptake and subcellular localization of AgNPs using transmission electron microscopy (TEM). AgNPs were internalized within 1 h and were found in membrane bound vesicles, likely endosomes, in all cell lines after 1 h exposure to AgNPs . The celTP53 (A549 and BEAS-2B), whereas in the TP53 null cell lines  the cells accumulated mainly in the G2 phase. The population of cells in S-phase decreased after ionizing radiation in all cell lines. On the other hand, the AgNP exposure induced G2 arrest in A549 and Calu-1 cell lines, S-phase arrest in BEAS-2B and did not seem to have any effect on the cell cycle in the NCI-H358 cells. Only in the Calu-1 cells the exposure to combined AgNPs and ionizing radiation appeared to have a statistically significant effect on the increase in cell accumulation in G2 phase (p = 0.037 for 1 μg/mL AgNPs with 2 Gy irradiation and p = 0.028 for 10 μg/mL AgNPs with 2 Gy irradiation as compared with AgNPs only).The cell cycle was studied with flow cytometry using PI-staining. Exposure to AgNPs and ionizing radiation affected the cell cycle in all the cell lines studied , but the2O2 detection and MitoSOX as a more general sensor of superoxide and mitochondrial redox state. The cell lines in our study had different basal levels of mitochondrial superoxide whereas the mitochondrial H2O2 levels were more similar, except for BEAS-2B that had the highest basal level of both H2O2 and superoxide . Exposure to AgNPs increased both mitochondrial H2O2 .The mitochondrial ROS was measured in live cells using flow cytometry. MitoPY1-probe was used for Hperoxide . The celial H2O2 a and mitial H2O2 b in all 2C containing a protein sulfenic acid trapping moiety for detecting oxidized proteins and a coumarin moiety for the localization into the mitochondria and fluorescence detection [Mitochondrial protein oxidation was analyzed using the recently developed probe DCP-NEtetection . In lineMitochondrial protein oxidation also increased in the AgNP-sensitive cell lines as a function of increasing concentrations of AgNPs, and the increased oxidation was statistically significant in particular for concentrations greater than 10 μg/mL b. The 2 2C labeling, followed by S-phase and G1 having the lowest amount of labeling. Nevertheless, the changes induced by the AgNP exposure on mitochondrial protein oxidation could be seen in all cell cycle phases and are, thus, not linked to the mitochondrial content of cell cycle phases. The same results were obtained for the other three cell lines (data not shown).To verify that the changes in the level of protein oxidation labeling were not due to changes in mitochondrial content induced by treatment with AgNPs, we used MitoTracker Green to quantify mitochondrial content in the cell. We did not detect any significant change with AgNP treatment in the four cell lines studied here e. HoweveThe mitochondrial respiration was evaluated with the Seahorse MitoStress assay  after 1 Overall, while there appeared to be some increase in the basal respiration, proton leak and decrease in spare respiratory capacity and coupling efficiency with the AgNP treatment, these changes were not statistically significant d. Howevep = 0.019, BEAS-2B: p = 0.0.10, NCI-H358: p = 0.011).Thus, despite the changes in mitochondrial ROS and protein oxidation induced by AgNP treatment, mitochondrial respiration and ATP production/coupling efficiency were not impaired by treatment. In order to account for non-mitochondrial effects on ATP levels, we also measured the ATP content in the cells using Promega CellTiter Glo-assay . Treatme+ released from the nanoparticles, induce ROS and disrupt mitochondrial function [Several recent studies have shown that exposure to AgNPs is cytotoxic in different cell lines, but with variable response ,39,40,43function ,5,25,44.function ,45.The studies presented here were designed to mimic potential environmental exposure to AgNPs. In this case, both intracellular and extracellular exposure to AgNPs can have effects. Thus, all cell lines were exposed to equivalent doses of AgNPs, in equivalent volumes of culture media, and cells were plated in identically sized wells at similar cell density, ensuring comparable exposure to AgNPs. Furthermore, the data were collected at multiple time points and doses of AgNPs to be able to assess differences in the timing, magnitude, and type of responses detected among cell types.We initially evaluated the AgNP-induced cell toxicity using a battery of tests starting with the effects on cell proliferation. As others have reported in various cell lines ,5,15,43,TP53, and decrease of cells in the S-phase in all cell lines. Interestingly, the effects of ionizing radiation on cell cycle seemed to be different from the AgNP exposure, and there were no combined effects, even though in addition to DNA damage, ionizing radiation can also induce mitochondrial dysfunction and increase in cellular and mitochondrial ROS [AgNPs have been shown to induce DNA damage, cell death by apoptosis and necrosis, and also cell cycle arrest in different cell lines . The effrial ROS ,55.2O2 and MitoSOX as an indicator of superoxide and other ROS. Both of them showed an increase in fluorescence after 24 h exposure to AgNPs in the AgNP-responsive cell lines , but not in the AgNP-tolerant NCI-H358 cell line. In line with these results, there was also a time- and dose-dependent increase in the mitochondrial protein oxidation in the AgNP-sensitive cell lines, but not in the NCI-H358 cells, where a slight decrease was observed at higher concentrations of AgNPs. The TEM imaging showed internalized AgNPs in all cell lines after 1 h exposure, but at higher level in the AgNP-sensitive BEAS-2B and Calu-1 cells, correlating with the stronger increase in protein oxidation (occurring at earlier time points and at lower doses of AgNPs) compared with A549 and NCI-H358 cells. Interestingly, ionizing radiation increased mitochondrial superoxide and protein oxidation only in the A549 cells, which was not further increased with exposure to AgNPs. The different phases of the cell cycle have different redox states [Since the mitochondria appear to be an important target for the effects of AgNPs, we wanted to analyze the redox state of mitochondria by studying both the mitochondrial ROS and protein oxidation. We used two mitochondrial fluorescent probes to study ROS, MitoPY1 for Hx states ,56, and x states . In our We also analyzed the effects of AgNPs on mitochondrial respiration using Seahorse MitoStress assay. The AgNP-responsive cell lines had higher spare respiratory capacity both in the absence and presence of AgNPs, possible due to higher mitochondrial content, than the tolerant NCI-H358. Interestingly, Calu-1, the cell line most sensitive to AgNPs had also the lower capacity for ATP-production. The mitochondrial spare respiratory capacity seemed to decrease slightly with the AgNP treatment along with an increase in the proton leak pointing to mitochondrial dysfunction, whereas the basal respiration appeared to increase. In our study, the changes in the basal respiration, spare respiratory capacity, coupling efficiency or other key parameters of mitochondria function induced by AgNPs were not statistically significant in any of the four cell lines investigated. The effects of AgNPs on mitochondrial respiration have been little studied, but there has been at least one report also showing increased oxygen consumption after exposure to AgNPs in murine hippocampal neuronal HT22 cells [+ rather than direct interaction of AgNPs with mitochondria. There was only a small additional decrease in ATP with longer exposure to AgNPs, except for Calu-1 which showed almost complete loss of ATP and cell viability after 24 h. Interestingly, a similar decrease in ATP was observed in NCI-H358 cells and yet this did not impact its cellular proliferation. Other studies have also shown a decrease in ATP levels after exposure to AgNPs and loss of mitochondrial membrane potential . It While the clinical significance of our findings remains to be established, the data are consistent with the pathology of exposure to AgNPs. AgNPs are known to penetrate the lung structures deep into the alveoli through diffusion and to cross the blood-air barrier of the lungs leading to lung inflammation and fibrosis, as well as toxicity to other organs  . Interes+, intracellular relay of redox signaling from endosomes to mitochondria, and the intrinsic capacity of individual cell lines to interfere with this relay and/or respond to mitochondrial oxidative challenge. We have shown that exposure to AgNPs decreases cell proliferation and induces cell cycle arrest in A549, BEAS-2B and Calu-2, but not in NCI-H358 cells. The AgNPs increase mitochondrial ROS and protein oxidation in time- and dose-dependent manner in the sensitive cell lines, but without altering the mitochondrial respiration significantly. The change in mitochondrial redox state was accompanied by a decrease in cellular ATP levels, including in the NCI-H358 cells resistant to AgNPs treatment. The more sensitive cell lines had more mitochondrial content and spare respiratory capacity, potentially making them more susceptible to mitochondrial damage leading to increased ROS levels. Interestingly, ionizing radiation seemed to have separate effects from AgNP exposure, and there was no interaction with AgNPs in most cell lines with the exception of NCI-H358 which showed decreased proliferation with combined radiation and AgNPs treatment at higher doses of radiation.The results of the studies presented here reveal a broad variability in the sensitivity of lung cells to AgNPs determined by several contributing processes: cellular uptake of AgNPs and conversion to Ag