This library of compounds includes [(C^N^C)PtII(L)] (L=dimethylsulfoxide (DMSO) 2 and triphenylphosphine (PPh3) 3) and [((C^N^C)PtII)2(L‘)] (where L‘=N‐heterocycles (pyrazine (pyr) 4, 4,4‘‐bipyridine  5 or diphosphine butane (dppb) 6). Their cytotoxicity was assessed against four cancerous cell lines and one normal cell line, with results highlighting significantly increased antiproliferative activity for the dinuclear complexes (4–6), when compared to the mononucleated species (2 and 3). Complex 6 is the most promising candidate, displaying very high selectivity towards cancerous cells, with selectivity index (SI) values >29.5 (A2780) and >11.2 (A2780cisR), and outperforming cisplatin by >4‐fold and >18‐fold, respectively.A series of cyclometallated mono‐ and di‐nuclear platinum(II) complexes and the parent organic ligand, 2,6‐diphenylpyridine  A study of the cytotoxicity of a library of cyclometallated mono‐ and di‐nuclear platinum(II)‐complexes, against a range of ovarian and breast cancer cell lines, identifies the importance of both ligand type and nuclearity on their antiproliferative activity. The lead candidate, a di‐nuclear platinum(II)‐phosphine complex, exhibits very high selectivity towards cancerous cells, with a selectivity index (SI) value >11.2 against cisplatin‐resistant ovarian cancer cells and outperforming monodentate analogues by >11‐fold and cisplatin by >18‐fold. Since the approval of the chemotherapeutic drug cisplatin, by the FDA in 1978, platinum(II)‐based complexes have become integral in the clinical treatment of a range of different cancers.Cyclometallated platinum(II) complexes have emerged as attractive alternatives to existing clinical antiproliferative platinum drugs, and several compounds were reported to possess moderate to potent cytotoxicity against cancer cell lines that are resistant to current platinum(II) anticancer drugs.5, 6 TN‐heterocycles and thiolates in the fourth coordination site, against a panel of human cancer cell lines including two human ovarian carcinomas, A2780 and A2780cisR.II(L)], wherein C^N^C is a tridentate dianionic cyclometallating motif bearing a range of aryl groups; including phenyl, naphthyl and dibenzoacridine derivatives and L=DMSO or acetonitrile (MeCN), displayed good to moderate cytotoxicity against human cell lines; colorectal adenocarcinoma (HT‐29) and breast adenocarcinoma (MDA‐MB‐231).Early reports from Lowe and co‐workers outlined the potent cytotoxicity of cyclometallated platinum(II)‐complexes incorporating a N^N^N terpyridine (terpy) ligand scaffold, and a range of 2(μ‐NHC)]+ platinum(II) complexes where HC^N^N=6‐phenyl‐2,2‘‐bipyridyl and μ‐NHC=a bridging N‐heterocyclic carbene ligand. They showed that through increasing the length of the linker between the two metal centres, up to a 2‐fold increase in cytotoxicity was induced against a panel of human cell lines, including cervix epithelioid adenocarcinoma (HeLa), liver hepatocellular carcinoma (HepG2) and nasopharyngeal carcinoma (SUNE1).+ displayed significantly higher antiproliferative activity, with nanomolar to sub‐micromolar potency (IC50=0.057–1.3 μm), compared to the dinuclear [((C^N^N)Pt)2(μ‐NHC)]+ platinum(II) complexes, (IC50=3.9–9.4 μm) against three tested cancer cell lines .2(bipy)] (where N^N^N=terpy and bipy is a range of pyridine substituted derivatives connected by different linker groups) against several human ovarian cell lines including those resistant to cisplatin and doxorubicin; CH1, CH1cis, CH1dox, A2780 and A2780cisR. Generally, they found that the presence of short rigid linkers, for example, 4,4‘‐bipyridine , between the metal centres generated complexes with potent cytotoxicity against the tested cancer cell lines including nanomolar IC50 values against the doxorubicin resistant ovarian cell line CH1dox.Polynuclear platinum(II) complexes represent an important growing class of anticancer agents with potential clinical significance.24, 25II(L)] (L=dimethylsulfoxide (DMSO) 2 and triphenylphosphine (PPh3) 3) and [((C^N^C)PtII)2(L‘)] (L‘=N‐heterocycles (pyrazine (pyr) 4, 4,4‘‐bipyridine  5 or diphosphine butane (dppb) 6) respectively, (where HC^N^CH=diphenylpyridine), against a range of cancer cell lines. This series of cyclometallated platinum(II) complexes were designed to consider the influence of three important structural features; 1) changes to the nature of the donor atom at the fourth coordination site of the metal centre  , 2) size of ligand spacer between the two metal centres for two dinuclear complexes (from pyrazine (pyr) to 4,4‘‐bipy) and 3) nuclearity of the complex (from mono‐ and di‐nuclear). We also report chemosensitivity studies against a normal cell type, showing that the most promising candidate, [((C^N^C)PtII)2(dppb)], is non‐toxic towards normal cells, unlike cisplatin, which demonstrates high cytotoxicity.In this study, we investigate the SARs for a series of mono‐ and di‐nuclear cyclometallated C^N^C platinum(II) compounds Figure ,  2 (where DMSO=dimethylsulfoxide) and [(C^N^C)Pt(PPh3)] 3 (where PPh3=triphenylphosphine), and three dinuclear platinum(II) complexes [((C^N^C)Pt)2(pyr)] 4, [((C^N^C)Pt)2] 5 and [((C^N^C)Pt)2(dppb)] 6  and is due to the two phosphines coupling with the platinum centres in this symmetric molecule.The library of compounds consists of a ligand, 2,6‐diphenylpyridine ocedures.33, 346 suitable for X‐ray diffraction were obtained through the slow evaporation of a concentrated chloroform solution at room temperature. The complex crystallizes in a monoclinic crystal system and solution refinement was performed in the space group P21/c (Table S3 in Supporting Information). The molecular structure of 6 is shown in Figure pseudo square‐planar geometry expected of d8 PtII complex (Tables S4 and S5).6, there are intermolecular, edge‐to‐face, π–π stacking interactions present between the phenyl rings of dppb and aromatics of the C^N^C pincer ligand .Bright yellow crystals of 1, complexes 2–6, cisplatin (CDDP), oxaliplatin (OXA) and carboplatin (CARB) were all screened for their cytotoxicity against human cell lines: cisplatin‐sensitive ovarian carcinoma (A2780), cisplatin‐resistant ovarian carcinoma (A2780cisR) and breast adenocarcinomas (MCF‐7 and MDA‐MB‐231). The IC50 values were obtained via the MTT assay after a 96 h incubation period of each compound with the cells at 37 °C and 5 % CO2  complexes, 2 and 3, have significant differences in their cytotoxicity. The potency of complex 2 increases by up to 10‐fold against MCF‐7 in comparison to ligand 1 (p<0.05), and exhibits similar potency (IC50=4.4±0.5 μm) to CDDP (IC50=3.07±0.02 μm) against MDA‐MB‐231 (p=0.01). Importantly, 2 is the only compound in this library which has noticeable toxicity towards MCF‐7 (IC50=10±1 μm), however, it remains >6‐fold less cytotoxic than CDDP (p<0.05). A recent report by Klein and co‐workers determined that 2 has a IC50 value against MDA‐MB‐231 of 12.12±1.84 μm, which is within experimental error.2 is more active towards the cisplatin‐resistant ovarian cell line A2780cisR than the parental cisplatin‐sensitive A2780 cells. On replacing the monodentate DMSO ligand in 2 for PPh3 in 3, the activity significantly decreases, and 3 is non‐toxic against all cell lines tested (IC50>100 μm) (Table Ligand 4 was designed to study the cytotoxicity effects of a dinuclear system with the short pyrazine linker. The cytotoxicity of 4 increases by up to 27‐fold and 5‐fold against A2780 (p<0.05), in comparison to mononuclear complexes 2 and 3, respectively. Upon extending the linker to 4,4‘‐bipy (5), the compound remains equitoxic to that of 4 against A2780, however, the toxicity decreases against all other cell lines, with up to a 2‐fold decrease against A2780cisR and an 11‐fold decrease against MDA‐MB‐231 (p<0.05). Harper et al. showed that the related [((N^N^N)Pt)2] (N^N^N=terpy) analogue of 5 has been reported to have GI50 values of 2.7±1.3 μm against A2780 and 5.6±0.4 μm against A2780cisR.50 values of >25 μm against A2780 and 9.6 μm against A2780cisR.50 values of 5 and the [((N^N^N)Pt)2] (N^N^N=terpy) analogue, highlights that the nature of the pincer ligand is also an important factor on determining the cytotoxicity of these cyclometallated platinum(II) complexes. On modification of the linker to a flexible butyl substituent, in the dinuclear diphosphine complex 6, the cytotoxicity against MDA‐MB‐231 decreases even further, with >4‐fold decrease when compared to 5 and >52‐fold decrease when compared to 4. Moreover, increasing the nuclearity of the platinum(II)‐phosphine compounds from mononuclearity in 3 to dinuclearity in 6 results in significantly increased cytotoxicity values against the ovarian carcinoma cell lines, with up to >29‐fold and >11‐fold increases observed against A2780 and A2780cisR, respectively. As the IC50 values of 3 against both ovarian cancer cell lines, A2780 and A2780cisR, is greater than the tested threshold concentration of 100 μm, this observed increase in cytotoxicity between these two platinum(II)‐phosphine ligands could be greater than reported here. As for 3, complex 6 is non‐toxic against MCF‐7 and MDA‐MB‐231 (IC50>100 μm).Complex 2 increasing the potency in all test cancer cells lines by up to 25‐fold compared to the analogous phosphine‐platinum(II) complex 3 , ii) the addition of a second platinum centre and linker unit increases the potency of the compounds by up to 29‐fold (e.g. 3 versus 6 against A2780), iii) the shorter pyrazine linker in complex 4 is the optimal linker of the N‐heterocyclic dinuclear species exhibiting up to sub‐micromolar potency against MDA‐MB‐231 (IC50=1.9±0.5 μm). A notable result is that of complex 6, which is non‐toxic towards either of the two human breast cancer cell lines , has significantly increased cytotoxicity against the human ovarian cancer cell lines, >29‐fold against A2780  and >11‐fold against A2780cisR .On analysis of these results, no definitive SARs can be determined, however, some important structural features can be highlighted; i) the nature of the ligand plays a significant role in determining the cytotoxicity of the mononuclear complexes, with the presence of the sulfoxide ligand in CDDP, OXA and CARB), which exhibit high potency towards normal cell types, this library of compounds was screened against normal prostate cell line, PNT2 , 8.5±0.4 μm (CDDP) and 27±2 μm (CARB). Ligand 1 and complex 3 are non‐toxic towards PNT2 (IC50 >100 μm). Complexes 2, 4 and 5 all remain moderately cytotoxic against the normal cell line, and even though complex 4 is >4‐fold more cytotoxic against MDA‐MB‐231 than PNT2, it remains relatively cytotoxic against normal cells (IC50=8.3±0.6 μm). The most promising result is observed for 6, which remains non‐toxic against PNT2 (IC50>100 μm) yet is cytotoxic towards human ovarian carcinomas.Due to the current issues of the clinical platinum compounds  and 6.7‐fold (A2780cisR) , however, 6 exhibits a SI value of 29.5, which is 4‐fold higher than that of CDDP (p<0.05). Importantly, the SI values observed for complex 6 against A2780cisR show a higher degree of selectivity than the clinical platinum compounds, with SI values >18‐fold (CDDP and OXA) and >42‐fold (CARB).The selectivity index (SI) values were calculated for all compounds, using the ICne Table . Whereby1–6 to target the ovarian cisplatin‐resistance cell line A2780cisR, the IC50 values were compared with those from the ovarian cisplatin‐sensitive cell line A2780. Resistance factors (RF)were obtained by dividing the IC50 values of A2780 by A2780cisR, where values >1 indicate a preference for the cisplatin resistant cell line A2780cisR. The RF values could not be calculated for ligand 1 and complex 3, as IC50 values are >100 μm against both ovarian cell lines. As with the clinical platinum drugs, complexes 4–6 are all more cytotoxic towards A2780. However, complex 2 has a slightly higher selectivity for A2780cisR, with a RF value of 1.4 .As many cancers become resistance to drugs, including the clinical platinum drugs, there is an urgent need to address the issues of drug resistance, by designing new and effective drugs. To address the potential of compounds 1–6 in comparison with the clinical drugs, the sensitivity factors (SF) were calculated by dividing the IC50 values of the clinical drugs by the IC50 values of compounds 1–6. An SF >1 indicates a selectivity for our library of compounds over the clinical drugs  and A2780cisR (4 and 6) whilst the rest of the library displays SF<1 against the other tested cell lines. When the biological performance of 1–6 is compared to OXA  are the most complex and aggressive types of breast cancer,2, 4–6 all have increases activity towards one or more of the ovarian carcinomas, when compared to CARB, with significantly higher activity against A2780 displaying SF ranging from 4.3 (5) to 5 (6) and, notably, against the cisplatin‐resistance ovarian cell line A2780cisR, with SF ranging from 5.4 (5) to 11.4 (4). Additionally, complex 2 has significantly higher activity when compared to CARB against the breast adenocarcinoma MCF‐7, displaying an IC50 value at least 10x higher than this clinical drug.To address the activity of compounds P Figure , complexA Figure , only cos Figure . In part4 and 6. Although complex 4 is moderately cytotoxic towards the normal cell line, its increased selectivity towards the TNBC line MDA‐MB‐231 is promising and should be investigated in further studies. Complex 6 is non‐toxic towards the normal cell line and has the highest cancer cell selectivity for this library, warranting further modifications and in‐depth in vitro studies. On analysis of these results, it is possible that these complexes have mechanism of actions which differs from that of the clinical platinum drugs. As it has previously been shown that cyclometallated N^N^N platinum(II) cationic complexes form strong intercalations with DNA, with binding constants from 0.8×105 m−1 to 2.0×107 m−1, and [((C^N^C)PtII)2(L‘)] respectively, where H C^N^CH is 2,6‐diphenylpyridine, L is a monodentate ligand (DMSO 2 or PPh33) and L‘ is a bidentate ligand . All compounds were screened against human ovarian carcinomas (A2780 and A2780cisR), human breast adenocarcinomas (MCF‐7 and MDA‐MB‐231) and normal prostate cell line (PNT2). There are no definite conclusions which can be drawn on the SARs however, some general trends can be observed.In conclusion, we have synthesized a library of mono‐ and di‐nuclear, cyclometallated platinum(II) complexes, of the general formula, + (100 %); HR‐ESIMS (+ve): calcd 232.1121 found 232.1115 for C17H14N. Elemental Analysis: Anal. Found: C: 88.2, H: 5.7, N: 6.1 %. Anal. Calculated: C: 88.3, H: 5.7, N: 6.1 %.2: To a solution of H2L  in acetic acid (55 mL) was added a solution of K2PtCl4  in H2O (3 mL) in a dropwise manner to generate a light rose coloured solution. The mixture was refluxed for 18 h, after which time, the red colour of the Pt salt had disappeared. The yellow precipitate that had formed was filtered off and washed with H2O (4 mL), acetone (4 mL), Et2O (4 mL) and pet. ether (2 mL). The yellow solid was then dissolved in DMSO (3 mL) and K2CO3  and H2O (2 mL) were added and the mixture heated to 90 °C for 1 h. After this time, the reaction mixture was allowed to cool to room temperature and upon the addition of H2O (10 mL), the product precipitated out as a bright yellow solid. This crude product was subjected to flash column chromatography (CH2Cl2). Single bright yellow crystals suitable for X‐ray diffraction were grown through the slow evaporation of chloroform from a saturated solution of 2. . 1H NMR : δ 7.81 =29.6 Hz, ArH), 7.63 , 7.49 , ArH), 7.33–7.27 , 7.13 , 3.69 =27.2 Hz, 2 × CH3); IR (solid) νmax 3034 (w), 2920 (w), 1598 (m), 1577 (m), 1562 (m) cm−1; LR‐ESIMS (+ve) m/z (%): 503 [M+H]+(100 %); HR‐ESIMS (+ve): calcd 503.0751 found 503.0745 for C19H18NO2NaPtS. Elemental Analysis: Anal. Found: C: 45.6, H: 3.3, N: 2.8 %. Anal. Calculated: C: 45.4, H: 3.4, N: 2.8 %.3: To a solution of 2  in CH2Cl2 (2 mL) was added PPh3  and the resultant pale‐yellow solution was stirred at ambient temperature for 1 min. After this time, the excess solution was removed under reduced pressure and the resulting yellow residue was subjected to flash column chromatography  to yield a bright yellow solid. Single crystals suitable for X‐ray crystallography were grown through the slow diffusion of di‐ethyl ether into a saturated solution of 3 in chloroform. . 1H NMR : δ 7.94 , 7.85–7.80 , 7.73 , 7.62 , 7.60–7.51 , 6.93 , 6.62 , 6.18 =32.4 Hz, ArH); IR (solid) νmax 3040 (w), 1597 (m), 1563 (m), 1508 (w) cm−1; LR‐ESIMS (+ve) m/z (%): 687 [M+H]+(100 %); Elemental Analysis: Anal. Found: C: 61.1, H: 3.7, N: 2.1 %. Anal. Calculated: C: 61.2, H: 3.8, N: 2.0 %.4: To a solution of 2  in CH2Cl2 (2 mL) was added pyrazine  and the resultant pale‐yellow solution was stirred at ambient temperature overnight. After this time, the red precipitate that had formed was filtered off, washed with CH2Cl2 (2 mL) to give the desired product as a bright red solid. . 1H NMR : δ 8.65 , 7.84 , 7.76 , 7.65–7.63 , 7.19 , 7.08 ; IR (solid) νmax 3043 (w), 1697 (w), 1598 (m), 1575 (m), 1560 (m) cm−1; LR‐ESIMS (+ve): 929 [M+H]+ (100 %); Elemental Analysis: Anal. Found: C: 49.0, H: 2.8, N: 6.0 %. Anal. Calculated: C: 49.1, H: 2.8, N: 6.0 %.5: To a solution of 2  in CH2Cl2 (2 mL) was added 4,4‘‐bipyridine  and the resultant pale‐yellow solution was stirred at ambient temperature overnight. After this time, the red precipitate that had formed was filtered off, washed with CH2Cl2 (2 mL) to give the desired product as a bright orange solid. . 1H NMR : δ 8.73 , 7.84–7.82 , 7.77 , 7.66–7.63 , 7.20 , 7.08 ; IR (solid) νmax 3041 (w), 1597 (m), 1575 (m), 1558 (m), 1541 (m) cm−1; LR‐ESIMS (+ve): 1005 [M+H]+; Elemental Analysis: Anal. Found: C: 51.9, H: 3.0, N: 5.4 %. Anal. Calculated: C: 52.6, H: 3.0, N: 5.6 %.6: To a solution of 2  in CH2Cl2 (2 mL) was added dppb  and the resultant pale‐yellow solution was stirred at ambient temperature for 1 h. After this time, the pale‐yellow precipitate that had formed was filtered off and yellow crystals suitable for X‐ray diffraction were grown through the slow diffusion of pet. ether into a saturated solution of chloroform . m.p. 310–311 °C; 1H NMR : δ 7.70–7.65 , 7.44 , 7.38–7.28 , 6.93 , 6.70 , 6.55 , 2.62–2.55 , 2.01–1.99 ; 13C{1H} NMR : δ 165.7 (JC‐Pt=104 Hz), 150.2, 139.6, 138.0 (JC‐Pt=116 Hz), 132.6, 132.5 (JC‐Pt=100 Hz), 129.5, 129.1, 127.5, 127.4, 123.2, 122.7, 114.2, 25.7, 25.3; 31P NMR : δ 20.3 (JP‐Pt=4960 Hz); IR (solid) νmax 3047 (w), 2936 (w), 2918 (w), 1597 (m), 1578 (m), 1565 (m), 1546 (m); LR‐ESIMS (+ve): 1275 [M+H]+; 1297 [M+Na]+; HRESI (+ve): calcd 1275.2823 found 1275.2803 for C62H51N2P2Pt2; calcd 1297.2642 found 1297.2637 for C62H50N2NaP2Pt2.Deposition Number(s) 1998373, 1998377 and 1998378 contain(s) the supplementary crystallographic data for this paper. These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service www.ccdc.cam.ac.uk/structures.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


In vitro cancer drug testing carries a low predictive value. We developed the human leiomyoma–derived matrix “Myogel” to better mimic the human tumor microenvironment (TME). We hypothesized that Myogel could provide an appropriate microenvironment for cancer cells, thereby allowing more in vivo–relevant drug testing. We screened 19 anticancer compounds, targeting the epidermal growth factor receptor (EGFR), MEK, and PI3K/mTOR on 12 head and neck squamous cell carcinoma (HNSCC) cell lines cultured on plastic, mouse sarcoma–derived Matrigel (MSDM), and Myogel. We applied a high-throughput drug screening assay under five different culturing conditions: cells in two-dimensional (2D) plastic wells and on top or embedded in Matrigel or Myogel. We then compared the efficacy of the anticancer compounds to the response rates of 19 HNSCC monotherapy clinical trials. Cancer cells on top of Myogel responded less to EGFR and MEK inhibitors compared to cells cultured on plastic or Matrigel. However, we found a similar response to the PI3K/mTOR inhibitors under all culturing conditions. Cells grown on Myogel more closely resembled the response rates reported in EGFR-inhibitor monotherapy clinical trials. Our findings suggest that a human tumor matrix improves the predictability of in vitro anticancer drug testing compared to current 2D and MSDM methods. Current preclinical anticancer drug testing carries a low predictive value, since only 5% of compounds showing efficacy in in vitro tests are licensed following clinical trials . TherefoHead and neck squamous cell carcinoma (HNSCC) represents the sixth most common cancer worldwide, with a relatively low survival rate of around 50%  and its In this study, using a high-throughput drug screening method, we tested 12 HNSCC cell lines cultured on plastic, mouse sarcoma–derived Matrigel, or Myogel using 19 anticancer compounds targeting EGFR, MEK, and PI3K/mTOR. We compared the efficacy of the anticancer drugs tested in vitro against the patients´ response rates in 19 monotherapy clinical trials. We identified different drug effects between Myogel and the other testing conditions, and demonstrated that Myogel in in vitro drug screening replicates the best results from clinical trials. The cell viability readout revealed differential EGFR- and MEK-inhibitor effects between Myogel and the other testing conditions . HoweverWe performed the Friedman test followed by a pair-wise comparison of drug responses under five culturing conditions , Myogel 3D, Matrigel 2D, and Matrigel 3D) for all cell lines in order to detect differences in drug efficacy . SignifiWe collected data from clinical trials on the anticancer compounds used as monotherapies for HNSCC patients until 17 March 2018 . Only ceThe response rate for each anticancer compound was calculated from our drug screen using DSS ≥ 5 as the cut-off point . We alsoRapamycin analog mTOR inhibitors, such as temsirolimus which is currently in a phase II trial, showed a high response rate, but a relatively low effect (0 ≥ DSS < 10) in each of our in vitro testing conditions . SeveralTraditionally, new anticancer compounds are tested on cancer cell lines cultured on top of plastic wells, followed by testing using xenograft mouse models prior to clinical trials . HoweverIn solid tumors, cancer cells are influenced by TME containing hundreds of effectors, such as stromal and immune cells, their cytokines, growth factors, and structural matrix proteins . ClearlyCurrently, cancer research is consistently moving towards 3D cell culture models for in vitro studies . Thus, sCell number measurement using imaging technique is not the optimal way to compare cell density in different matrices due to alternated cell morphology (spheroid in Matrigel and stellate-shaped in Myogel) and 3D growth . In thisThe EGFR inhibitors were considered promising anticancer drugs for HNSCC. However, only modest response rates have been found . Among an = 14) than in clinical samples (n = 55) . Ea. Ea23]. We designed different culturing conditions  and 1N sodium hydroxide were mixed with Myogel (500 µg/mL) to ensure complete gelation. For embedding, cells were first mixed with the matrix (Myogel + Collagen I or Matrigel) and cell suspension was added to the wells using an electronic pipette . After a 1-h incubation, a culture medium (10 μL per well) was added on top of the solidified matrix and the plates were returned to the incubator to sit overnight.Myogel and Matrigel were thawed overnight on ice (4 °C). We pre-chilled pipette tips and other equipment in a freezer (–20 °C) and 384-well plates on ice. Matrigel and Myogel were diluted with a cell culture media to 500 µg/mL. Matrices (5 µL) were added to 384-well plates using an electronic pipette  and the plates were centrifuged for 2 min at 300 rpm at 4 °C. Plates were left overnight in a cell culture incubator. In the control plates, only the cell culture media was added. On the following day, the cells were counted using the Scepter™ 2.0 Cell Counter  and suspended to 500 cells/well. The cell density was selected based on previous publication and optimized to avoid confluence on the last day of the experiment . To the We performed drug sensitivity and resistance testing (DSRT) on HNSCC cell lines cultured in different matrices. Each drug was tested over a 10,000-fold concentration range. We normalized the drug effect against positive (BzCl) and negative dimethyl sulfoxide (DMSO) control wells to calculate the dose–response curves for each drug in each cell line and matrices separately. To quantitatively profile alternate drug effects, we calculated the drug-sensitivity score (DSS) designed by the High Throughput Biomedicine Unit (HTB) at the Institute for Molecular Medicine Finland (FIMM). The DSS score has been described in several studies ,23,28, aOur DSRT protocol was adapted from a platform for leukemia cells used previously . DSRT cu® (CTG) 2.0 Luminescent Cell Viability Assay  was used to determine the efficacy of the drugs. We first imaged the plates using IncuCyte Live-Cell Imaging System  to observe cell density  on each drug comparing the five culturing conditions  to detect the differences in drug efficacy. Pair-wise comparisons were performed  applying the Bonferroni correction for multiple comparisons .https://clinicaltrials.gov) and PubMed. We included only completed clinical trials for drugs used as monotherapies for which the results were reported. We collected information on the objective response rates , the number of patients who received the monotherapy, and the number of responding patients in one table.Clinical trial data for 19 anticancer compounds in HNSCC patients were collected until 17 March 2018, from a website maintained by the National Library of Medicine , which revealed the differential EGFR- and MEK-inhibitor effects between Myogel and the other testing conditions (v/v) Brij 35; Sigma Aldrich) with a complete protease inhibitor cocktail  and PhosSTOPTM phosphatase inhibitors (Roche). After removing the cell debris by centrifugation, the protein concentrations were measured using a DC Protein assay . Next, 30 µg of a soluble protein were separated under reducing conditions using a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to an Immobilon P membrane . After blocking with the Odyssey® Blocking Buffer , the membrane was incubated with 1:1000 rabbit anti-EGFR , 1:1000 rabbit anti-p44/42 MAPK , 1:2000 rabbit anti-Phospho-p44/42 MAPK  or 1:2000 mouse anti-β-Actin  antibodies overnight at +4 °C [®20 (0.05%), 1:10000 IRDye® 680RD Goat anti-Rabbit IgG or IRDye® 800CW Goat anti-Mouse IgG secondary antibody (Licor Biosciences) was added for 50 min at room temperature and the membrane was washed three times for 5 min each. The Odyssey scanner (LI-COR Biosciences) was used to image the membrane and we used the Fiji software [To examine protein expression, 7.5 × 10nditions . The celat +4 °C . After wOur findings taken together detected varying drug effects between the human tumor–derived matrix Myogel compared to current 2D and MSDM methods. We found that Myogel in in vitro drug screening replicates the best results from clinical trials. We suggest that using the human tumor matrix in preclinical studies will diminish the number of false-positive preclinical results, reduce the cost of drug development, and minimize the unnecessary suffering of cancer patients.


Chinese hamster ovary cell lines, also known as CHO cells, represent a large family of related, yet quite different, cell lines which are metabolic mutants derived from the original cell line, CHO-ori. Dihydrofolate reductase-deficient DXB-11 cell line, one of the first CHO derivatives, serves as the host cell line for the production of therapeutic proteins. It is generally assumed that DXB-11 is identical to DUKX or CHO-DUK cell lines, but, to our knowledge, DXB-11 karyotype has not been described yet.Using differential staining approaches , we presented DXB-11 karyotype and revealed that karyotypes of DXB-11 and CHO-DUK cells have a number of differences. Although the number of chromosomes is equal—20 in each cell line—DXB-11 has normal chromosomes of the 1st and 5th pairs as well as an intact chromosome 8. Besides, in DXB-11 line, chromosome der(Z9) includes the material of chromosomes X and 6, whereas in CHO-DUK it results from the translocation of chromosomes 1 and 6. Ag-positive nucleolar organizer regions were revealed in the long arms of chromosome del(4)(q11q12) and both chromosome 5 homologues, as well as in the short arms of chromosomes 8 and add(8)(q11). Only 19 from 112 (16.96%) DXB-11 cells display identical chromosome complement accepted as the main structural variant of karyotype. The karyotype heterogeneity of all the rest of cells  occurs due to clonal and nonclonal additional structural rearrangements of chromosomes. Estimation of the frequency of chromosome involvement in these rearrangements allowed us to reveal that chromosomes 9, der(X)t, del(2)(p21p23), del(2)(q11q22) /Z2, der(4) /Z7, add(6)(p11) /Z8 are the most stable, whereas mar2, probably der(10), is the most unstable chromosome. A comparative analysis of our own and literary data on CHO karyotypes allowed to designate conservative chromosomes, both normal and rearranged, that remain unchanged in different CHO cell lines, as well as variable chromosomes that determine the individuality of karyotypes of CHO derivatives.DXB-11and CHO-DUK cell lines differ in karyotypes. The revealed differential instability of DXB-11 chromosomes is likely not incidental and results in karyotype heterogeneity of cell population. Chinese hamster ovary cell lines known as CHO cells represent a large family of related, but quite different cell lines which are metabolic mutants derived from the original cell line, CHO-ori –3, by clThe unique plasticity of the CHO genome has made these cells the major mammalian host cells for manufacturing of protein pharmaceuticals. The most industrially relevant CHO cell lines are CHO-K1 , CHO-S , CHO-DXBThe undoubted advantages of immortalized CHO cells include a high proliferation rate, and the ability to adapt to genetic manipulations and culture conditions. At the same time, these properties are associated with an increased mutation rate, changes in the genome structure and DNA methylation pattern –9. The kKaryotype heterogeneity of the cell population can be studied with two main methods: conventional karyotyping  or fluorescence in situ hybridization with chromosome-specific painting probes . In the first case, karyotype analysis compares unique banding patterns of chromosomes and can provide comprehensive information regarding chromosome rearrangements in individual cells although it may fail to identify all chromosomal material. On the contrary, M-FISH analysis provides complete identification of chromosomal material, but intrachromosomal rearrangements such as deletions, inversions, duplications, isochromosomes remain invisible. In addition, unlike with banding, the number of cells that can be karyotyped by M-FISH method is limited. We chose analysis of G-banded chromosomes as a simple, reliable, and low-cost method which allows to establish the type and level of chromosomal changes in individual cells, and therefore to estimate true karyotype heterogeneity. Besides, G-banded karyotype may be important for establishing the origin of recombinant strains derived from certain host cells. Finally, karyotyping of CHO cell lines—original CHO , DG-44 , CHO-DUKDHFR) gene and missense mutation (T137R) in the second allele [DHFR made this cell line very useful for transgenesis with a functional DHFR gene. Further selection of recombinant cells in a medium containing methotrexate results in amplification of both DHFR and the gene of interest. Thus, the DXB-11 cell line is the host in relation to the producer strains obtained on its basis.The DXB-11 cell line is one of the first CHO derivatives, generated at Columbia University. This line was developed as a result of chemical mutagenesis followed by gamma irradiation and represents a radiation mutant with a deletion of one allele of the dihydrofolate reductase  from Columbia University, New York, USA, in 1984. Cytogenetic analysis of these cells has not been performed until now.Here, we describe the G-banded karyotype of DXB-11 cells and present our estimation of the instability of each chromosome. We have found out that DXB-11 karyotype differs from the karyotype of CHO-DUK cell line. Besides, assessment of frequency of chromosome participation in additional structural rearrangements (ASR) reveals differential instability of individual DXB-11 chromosomes. Comparative karyotype analysis of CHO cell lines, including CHO-DUK and our data on DXB-11, allows us to make a distinction between stable and variable CHO chromosomes.2. Cells were passaged twice a week at a ratio of 1:3 just as 90% confluence was reached. Cytogenetic analysis was performed at passage 8 over 30 days of cultivation after decryoconservation. Cells in the exponential growth phase were exposed to KaryoMAX™ Colcemide™ Solution in PBS  at a final concentration of 0.1 µg/mL for 1 h, trypsinized, washed with phosphate buffered saline (PBS), and treated with 0.075 M KCl at room temperature for 15 min. Then the cells were fixed in prefrozen (− 20 °C) methanol : acetic acid (3:1) solution three times for 20 min each. Chromosome spreads were obtained by dropping of cell suspension onto cold wet slides which were air dried after.DXB-11 cell line from Collection of Cell Cultures of Vertebrates  was examined. DXB-11 cells were adherently maintained in T-25 culture flasks  containing 5 mL F-12К  supplemented with 10% fetal bovine serum  at 37 °C in a humidified incubator with 5% COPreparations of metaphase chromosomes were baked at 60 °C for 16 h and G-banded by treating with 0.02% trypsin followed by staining with 2% Giemsa solution in phosphate buffer . To reveCricetulus griseus) chromosomes at 325-band [p value < 0.05 was considered statistically significant.The chromosomes were analyzed with Axio Scope.A1 microscope coupled to an image capturing system AxioCam Cm1  using a 100 × oil objective. The number of chromosomes in 100 G-banded metaphase spreads was determined. At least 35 C- and Ag-stained metaphase spreads per each technique were examined using a 100 × oil objective. Karyotyping of 112 cells was performed on Axio Imager A2 microscope  equipped with the Ikaros4 Karyotyping System  using a 63 × oil objective. Percent of polyploid cells was estimated by viewing of 1000 metaphase plates. The nomenclature for Chinese hamster  had the same karyotype accepted as the main structural variant (SVK), since it most accurately reflects the combination of normal and structurally rearranged chromosomes (q11q22). Chromosome inv(3)(p26q11) is an inverted chromosome 3 homologue q1q22. ChrC-bands were observed on the long arms of both chromosomes 1 and at the terminal region of the long arm of chromosome der3) . Chromosome der(6) can also be considered a stable chromosome because its involvement in ASR was compatible with that of chromosomes inv(3)(p26q11) and mar4 (p > 0.05), but lower as compared to other chromosomes (p < 0.05). On the contrary, the most variable was chromosome mar2 (p < 0.05), which showed both extra copy and deletions of repeated regions of the long arm , mar22—in 15 (13.39%), mar23—in 6 (5.36%), mar24—in 4 (3.57%), mar25—in 3 (2.68%) cells. The rest of chromosomes had approximately the same degree of instability (p > 0.05). However, instability of chromosome add(8)(q11) was lower as compared to chromosome mar2 (p < 0.05), comparable with the instability of chromosome mar1 (p > 0.05), and higher (p < 0.05) as compared to all other chromosomes.The most stable were chromosomes 9, der(X)t, del(2)(p21p23), del(2)(q11q22), der(4) and add(6)(p11), that were rarely involved in ASR p < 0.0. Chromos1 (q11) and mar2 in ASR is of special interest Fig. b, c. In 1 Fig. b. These pes Fig. b. In onearm Fig. c. Beside1) was registered in 7 from 112 cells (6.25%) without t. Rare clonal ASR observed in 3 from 112 cells (2.68%) was the deletion of chromosome 5 short arm (q11q12), chromosomes of the 5th pair, and chromosome 8 in clonal ASR was revealed. So, for chromosome del(4)(q11q12), balanced translocation of chromosomes 1 and del(4)(q11q12) confirmed by Ag-staining  (q11q12) short arm  was the complex interstitial deletion of the long arm, der(3)2%) Fig. h. The clarm Fig. i, or to arm Fig. j, which As a whole, the chromosome material of different DXB-11 cells was similar in spite of their karyotype heterogeneity.Like other CHO cell lines, DXB-11 cells were characterized by significant karyotypic heterogeneity. Apart from a small number of cells that had the same karyotype (main SVK), other variants of the DXB-11 karyotype were not identified. However, our analysis of ASR showed that individual chromosomes undergo rearrangements with different frequency. The revealed differential instability of DXB-11 chromosomes occurred to be in agreement with previous data  concerniCricetulus griseus) and were characterized by multiple structural rearrangements with partial loss of material of chromosomes X and 2.According to the first description of karyotype of CHO cell line later named CHO-ori , the modHere, we have performed a comparative karyotype analysis of CHO derivatives Table  in accordhfr−, CHO-DUK, DXB-11 and DG-44 karyotypes demonstrated the presence of one normal chromosome homologue of the 1th, 5th and 9th pairs, as well as abnormal chromosomes Z2, Z4, Z5, Z7, Z8 and Z13. Apparently these chromosomes represent the most stable (i.e. conserved) part of CHO karyotype (p21p23), del(2)(q11q22) /Z2, der(4) /Z7, and add(6) /Z8 do undergo ASR in DXB-11 cells to a lesser extent. Stability of chromosomes 1, 2, 5, 8, 9, Z4, Z5, Z7 and Z8 in CHO-K1 and DG-44 cells was demonstrated by molecular cytogenetic methods . StabiliThe variable part of CHO karyotype is represented by chromosomes X, 1 (2nd homologue), 5 (2nd homologue), 7, 8, 10, Z3, Z9, Z10, Z11, and Z12. Apparently rearrangements of these chromosomes determine genetic diversity and individuality of the karyotype structure of different CHO cell lines.dhfr−, CHO-DUK, DXB-11 and DG-44. Chromosome der(Z11) contains an additional chromosome material on the long arm in DG-44 cells only [For example, establishment of CHO-K1 cell line was accompanied by a decrease in number of chromosomes from 21 (CHO-ori) to 20 (CHO-K1), and rearrangements of chromosomes X, 10, and abnormal chromosomes of the 3rd (Z3), 7th (Z10) and 8th Z11) pairs , 33. Chr pairs , Reorganization of material of chromosomes X (Xq), the 7th chromosome pair 7 and Z10), and chromosome 10 allows to distinguish CHO-K1 cells both from CHO-ori and CHO-K1 cells cultured in different conditions , and chr, 31–33. Thus, different ability of DXB-11 chromosomes to undergo ASR is non-random and corresponds to individual chromosome instability in CHO cell lines. It has been suggested that some regions of the CHO genome are predisposed to structural variations . Apparendhfr− and CHO-DUK from CHO-K1 and DG-44 is a visible deletion in the short arm of chromosome 2, which is associated with the loss of DHFR gene at 2p23 [DGFR alleles in DG-44 cell line is not accompanied by a notable change of G-banding pattern of the short arm of chromosome 2 judging by karyotype image [A significant difference between the cell lines DXB-11, CHO at 2p23 . The remdhfr− and CHO-DUK as was believed previously [dhfr− [dhfr− [dhfr− cell lines despite the great karyotype diversity of DXB-11 cells.We have shown here that the CHO cell line DXB-11 is not identical to CHOeviously , 15, 20.y [dhfr− , 15. It y [dhfr−  demonstr− [dhfr− , 15 by tdhfr− are associated with chromosome rearrangements of the variable part of the CHO karyotype, namely, Xq, 1, 5, 8, 10, and Z9. It remains unclear whether DXB-11 and CHO-DUK cells are the result of the divergence of a single cell line cultured in different laboratories or these lines originate from the different experimentally obtained DHFR-deficient CHO cell clones [dhfr− cell lines may be considered as CHO quasispecies [Thus, it should be once again pointed out that the differences between DHFR-deficient cell lines DXB-11 and CHO-DUK/CHOispecies .n located in the pericentromeric regions of Cricetulus griseus chromosomes [The mechanisms of chromosomal/genomic instability of CHO cells remain poorly understood. According to our data, the breakpoints involved in formation of abnormal chromosomes in DXB-11 cells are often located in centromeric and pericentromeric regions Fig. . It has omosomes , 35. At omosomes , might aThe genetic diversity of CHO cell lines appears at the level of both the karyotype and the genome. At the karyotype level, it shows through complex rearrangements which involve different chromosomes with different frequency , thus determining karyotype heterogeneity. At the genome level, sequencing of different CHO derivatives demonstrates single nucleotide polymorphisms, short insertions and deletions (InDels), DNA copy number variations and structural changes which lead to an increase in mutation frequency, the loss of genes and heterozygosity. Besides, different regions of the genome may undergo structural variations and genetic imbalance to a different extent , 8, 17. DXB-11 and CHO-DUK cell lines differ in karyotypes. DXB-11 cell population is characterized by a limited number of cells with identical chromosome complement and a predominant number of cells with a wide spectrum of clonal and nonclonal additional structural chromosome rearrangements. The revealed differential instability of DXB-11 chromosomes is most likely not incidental. Seemingly, karyotype heterogeneity of CHO cell lines is determined by rearrangements of variable CHO chromosomes.


Though high radiation doses can be delivered to the tumors, complete remission is still rare. Radiosensitization prior to PRRT is therefore considered to be a promising strategy to improve the treatment effect. In this study, effect and mechanism of mTOR inhibitors were investigated in a comprehensive panel of five NEN cell lines , employing assays for cellular proliferation, clonogenic survival, cell cycle modification and signaling. mTOR inhibition lead to growth arrest with a biphasic concentration-response pattern: a partial response at approximately 1 nM and full response at micromolar concentrations (8–48 µM). All cell lines demonstrated elevated p70S6K phosphorylation yet also increased phosphorylation of counterregulatory Akt. The pulmonary NEN cell line UMC-11 showed the lowest induction of phospho-Akt and strongest growth arrest by mTOR inhibitors. Radiation sensitivity of the cells (50% reduction versus control) was found to range between 4 and 8 Gy. Further, mTOR inhibition was employed together with irradiation to evaluate radiosensitizing effects of this combination treatment. mTOR inhibition was found to radiosensitize all five NEN cells in an additive manner with a moderate overall effect. The radiation-induced G2/M arrest was diminished under combination treatment, leading to an increased G1 arrest. Further investigation involving a suitable animal model as well as radioligand application such as  Side effects were found to be mild (Peptide receptor radioligand therapy (PRRT) has evolved as an important second-line treatment option in the management of inoperable and metastatic neuroendocrine neoplasms (NEN). In one of the earliest studies, comprising 310 GEP-NET patients, treatment with The serine/threonine kinase mTOR  is a central integrator of environmental signals such as nutrients, growth factors and stress, coordinating cell growth and proliferation. Depending on the proteins it is interacting with, it can form the two complexes mTORC1 and mTORC2 which have their own distinct downstream effectors. Activated mTORC1 initiates protein translation by phosphorylation of S6K1  and 4EBP1, which in turn further engage S6 ribosomal protein and eIF4E. At the same time, activated S6K1 negatively regulates the PI3K-Akt pathway by inhibition of IRS. Furthermore, mTORC1 promotes cell cycle progression, inhibits autophagy and controls transcription and the DNA damage response. On the other hand, mTORC2 is in charge of cell survival, metabolism and the actin cytoskeleton .Dysregulated mTOR signaling has been demonstrated in various cancers, which made mTOR a promising target and facilitated the development of derivatives of rapamycin, a naturally occurring mTOR inhibitor. Two examples for FDA-approved rapalogs with improved pharmacological and solubility qualities are temsirolimus CCI-779) and everolimus (RAD001) . They fo79 and ev177Lu-DOTATATE) in 16 patients. Overall response was observed in 44% of patients, and the maximum tolerated dose for everolimus in combination was found to be 7.5 mg daily  according to the manufacturer’s protocol. Colorimetric changes were measured at 562 nm and the obtained OD values were used to interpolate protein concentrations from a standard curve prepared with BSA (0–10 mg/ml).Protein gel electrophoresis was performed according to standard protocols. Cell lysates were separated on discontinuous SDS polyacrylamide gels. 10 µg of protein were diluted in 5x sample buffer and water in 10 µl total volume, denatured for 5 min at 95°C and spun down shortly. Polymerized gels were assembled in a Mini-PROTEAN Tetra tank , the upper chamber filled with top buffer  and the samples loaded into the lanes. The lower chamber was filled with bottom buffer  and the gels run at 100 V. The Precision Plus Kaleidoscope Standard (Bio-Rad) was used as molecular weight marker.Electrophoretically separated proteins were transferred to nitrocellulose membranes by the wet blot technique. For this, polyacrylamide gel, membrane, fiber pads and filter paper were equilibrated in transfer buffer  for 15 min, stacked and locked into a cassette. The cassette was placed into a mini Trans-Blot tank together with an ice pad and a stir bar and filled with transfer buffer. Blotting was performed for 1 h at 100 V and constant 350 mA on a magnetic stirrer. Membrane was stained with ponceau S for assessment of homogenous protein transfer, destained with water and incubated in blocking buffer (5 % w/v nonfat dry milk in TBST) for 60 min. After a short wash with TBST, membrane was incubated with primary antibody overnight at 4 °C. Membrane was washed three times for 5 min with TBST, incubated with HRP-coupled secondary antibody diluted in blocking buffer for 60 min at room temperature and again, washed three times for 5 min with TBST. Chemiluminescence was induced by addition of 500 µl SuperSignal West Dura (Pierce ThermoFisher) substrate and captured with a VersaDoc imaging system (Bio-Rad). Protein signals were analyzed and quantified with Image Lab software (Bio-Rad).The human neuroendocrine cell lines BON and QGP-1 (pancreatic), LCC-18 (colonic), and H727 and UMC-11 (pulmonary) were cultured in RPMI 1640 containing 10% fetal calf serum in a humidified atmosphere at 37°C with 5% CO2. BON cells were a kind gift of Courtney Townsend . LCC-18 cells were kindly donated by Kjell Öberg . QGP-1 were obtained from the Japanese Collection of Research Bioresources, and H727 and UMC-11 from ATCC . Cells were cultured for no longer than 15 passages. All human cell lines have been authenticated by DSMZ  using STR profiling.Cells were seeded at densities of approximately 5%–10% in 96-, 12-, or 6-well plates and grown overnight. Substances were added in medium on top of the wells in double concentration for the indicated final concentrations. Irradiation was performed using an external caesium-137 source at a dose rate of 1 Gy/min and further incubation without medium change. For combination treatments, cells were incubated for 24 h with the respective substance before radiation or radioligand treatment was applied.Cell cycle analysis was performed on the basis of DNA content measurement by flow cytometry. The fluorescence intensity of DNA binding dyes such as propidium iodide is proportional to the DNA content of the cell, thereby allowing the discrimination of cells in sub-G1, G1, S, and G2/M cell cycle phases . Cells wCells were seeded in quadruplicates at a density of 5,000 cells in 50 µl medium per well in 96-well plates, grown overnight and treated as described. After 96 h, metabolic activity was determined by addition of 100 µl medium containing 0.4 mM of the redox indicator resazurin. Cells were incubated for 3–4 h and the resulting fluorescence was measured with an EnVision Multilabel Plate Reader . Subsequently, the supernatant was removed, cells were fixated with 4 % formaldehyde for 10 min, stained with DAPI (1 µg/ml in PBS/0.1 % Triton) for another 10 min and wells were covered with 80 µl PBS for image acquisition. Four fields per well were imaged in an IN Cell Analyzer 1000  using the 4x objective. Image stacks were analyzed and nuclei counted by IN Cell software. The values were averaged and normalized as percent of control treated with vehicle.In comparison to short term cell viability assays, the clonogenic survival assay evaluates the ability of single cells to reproduce and form colonies, so called clones. Cells were seeded in duplicates and a density of 5,000 cells in 500 µl medium per well in 12-well plates and treated as described. Cells were incubated without medium change for 1–2 weeks. Finally, colonies were fixed with 70 % ethanol for 10 min, stained with crystal violet solution  for another 10 min and carefully rinsed with tap water. Plates were dried overnight and digitized with an Odyssey infrared scanner . For quantification, images were analyzed using the ColonyArea plugin for ImageJ .via this link: https://doi.org/10.5281/zenodo.3922212.If not indicated otherwise, dose-response curves were plotted with GraphPad Prism 5.3 and the data fitted using nonlinear regression and the variable slope model (four-parameters). As x values, base 10 logarithms of doses or concentrations were entered. Statistical analyses were performed with the same software. The quantitative data for this study have been permanently published in a public repository accessible 50 values in the nanomolar and micromolar range, respectively (50 differed only slightly between cell lines and assays (around 1 nM), whereas the high micromolar IC50 demonstrated greater variation. Here, values ranged from 8 to 21 µM for temsirolimus and from 30 to 48 µM for everolimus. At nanomolar concentration, the inhibitors reduced cell viability by 20%–75%, with BON being the most resistant cell line (20%) and UMC-11 the most sensitive (75%). In contrast, when applying micromolar concentrations, all NEN cell lines eventually showed a complete loss of cell viability. These effects are independent of the SSTR2 expression status, as BON-SSTR2 and QGP1-SSTR2 cells show no significant differences under either temsirolimus or everolimus treatment , LCC-18 (from colon), and H727 and UMC-11 (both from lung). The cells were incubated with either mTOR inhibitor and two parameters of cell viability were determined 96 hours after the start of the incubation: metabolic activity and cell number. In both assays, temsirolimus and everolimus led to a biphasic inhibition of cell viability in all five NEN cell lines Figure 1To investigate the inhibitors’ impact on long-term cell survival and proliferation, clonogenic assays were performed. Both inhibitors led to similar concentration-response curves, with the most profound effect on UMC-11 and H727 cells Figure 2As mTOR inhibitors are known to inhibit cell cycle progression in the G1 phase, their effect in the NEN cell lines was investigated. Cell cycle analysis by flow cytometry was performed 24 h to 96 h after treatment. Within 24 h, temsirolimus induced a moderate increase of cells in the G1 phase Table 2.To investigate how key intracellular effectors were influenced by mTOR inhibitor treatment, all five NEN cell lines were analyzed using Western blot. p70S6 kinase, involved in cell growth and cell cycle progression, is known to be directly activated by mTOR. Consequently, mTOR inhibition should lead to decreased p70S6 kinase activation/phosphorylation. Indeed, this was found in lysates of all five NEN cell lines after incubation with 1 nM or 100 nM everolimus for 6 or 24 h Figure 4In summary, both mTOR inhibitors showed antiproliferative activity in all five NEN cell lines, caused by suppression of the mTOR signaling pathway and G1 cell cycle arrest. However, the cell lines differed in their reaction towards treatment, depending on counteracting regulation and the extent and duration of G1 cell accumulation. Whereas the rather resistant BON cells exhibited only a transient G1 arrest, but increased phospho-Akt levels, the most sensitive UMC-11 cells showed persistent G1 arrest and barely detectable phosphorylation of Akt.Before investigating combined mTOR inhibitor and radiation treatment, the sole impact of irradiation on NEN cells was assessed by applying a single dose of 0–10 Gy. Cell viability assays revealed a dose-dependent reduction of metabolic activity and cell number in all investigated NEN cell lines Table 3.The five NEN cell lines under investigation were pretreated with temsirolimus for 24 h, subsequently irradiated and incubated for another 96 h. As observed before, cell numbers declined when applying increasing doses of temsirolimus or radiation Figure 6In addition, clonogenic survival assays were included to evaluate the impact of the combined treatment over a longer period. For this, NEN cells were irradiated with 0–4 Gy after preincubation with temsirolimus. Combination of both interventions resulted in clearly impaired cell survival in comparison to the single treatments Figure 8The previous analysis of cell cycle distribution following the respective single treatments revealed increased cell accumulation in G1 after mTOR inhibitor treatment, or in G2/M after irradiation. Here, the combined effect of both treatments was evaluated by preincubating the respective cell lines with 1 µM temsirolimus for 24 h before irradiating them with 10 Gy. In all five NEN cell lines, mTOR inhibitor pretreatment resulted in a diminished radiation-induced G2/M arrest after 24 h Table 4.In summary, combination of mTOR inhibitor treatment and irradiation indicated superiority compared to the single treatments as proven by increased decrease of cell numbers and cell survival. Preincubation with temsirolimus abrogated the irradiation-induced G2/M arrest in all NEN cell lines under investigation.PRRT has become an essential treatment option for advanced NEN. It demonstrated increased progression-free survival when compared to chemotherapy, radiation, and intervention with somatostatin analogs (SSAs). Objective response rates range between 15 and 35 % . However50 values for both substances in all five cell lines. Whereas nanomolar concentrations led to a moderate antiproliferative effect, micromolar concentrations suppressed cell viability completely. This is in line with a report from Shor et al. that also described a low-dose and high-dose effect of temsirolimus in different cancer cell lines  differed between the investigated cell lines. Pulmonary UMC-11 cells were found to be highly drug-sensitive, as cell viability was reduced by 75% after treatment at nanomolar concentrations, whereas BON cells proved to be rather resistant (20 % reduction). Long-term clonogenic survival assays over up to 2 weeks confirmed these results.First, the impact of the two mTOR inhibitors temsirolimus and everolimus on cellular processes was validated for the utilized NEN cell lines. Proliferation assays revealed a biphasic inhibition of metabolic activity and cell number, with two resulting ICll lines . In addiSignaling and cell cycle analysis were conducted to further elucidate the observed response in these cell lines. Similar to a study by Hurvitz et al. in breast cancer cell lines , inhibitCell cycle checkpoints at G1/S or G2/M phase transitions are important control mechanisms to maintain genomic integrity within the cell in response to environmental stress and DNA damaging agents such as irradiation. Their activation is mediated by a complex signaling network including cyclins, cyclin-dependent kinases and the key modulator p53, which is primarily involved in G1 cell cycle arrest . In the in vitro and further complements the reported results for pancreatic neuroendocrine BON and QGP-1 cells , preferentially SSTR2. However, for all NEN cell lines under investigation in this study, we have previously demonstrated low target expression and a lack of binding of the SSA octreotide. All five cell lines cells showed 10- to 1000-fold lower SSTR2 mRNA levels than human NEN tissues  have been characterized with regards to their genetic profile and have been shown to feature mutations untypical of neuroendocrine tumors or even neuroendocrine cancers   , 49. Whi3, TSC2)  or NT3  , primary in rats . In addihttps://doi.org/10.5281/zenodo.3922212.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Zenodo CG and VP designed and supervised research. SE and GT performed research and analyzed data. SE and CG wrote the paper. All authors contributed to the article and approved the submitted version.This work was supported by grants from the German Ministry of Education and Research (BMBF IP614 and IPT614A to CG). SE was the recipient of a fellowship by Sonnenfeld-Stiftung. Funding agencies had no influence on design, data acquisition of evaluation in any part of this study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lister strain of VACV, which encodes the enzyme super cytosine deaminase (SCD) that converts the prodrug 5-fluorocytosine (5-FC) into the chemotherapeutic compound 5-fluorouracil (5-FU). After treatment with GLV-1h94 alone, only half of the cell lines were defined as highly susceptible to GLV-1h94-induced oncolysis. When adding 5-FC, 85% of the cell lines became highly susceptible to combinatorial treatment; none of the tested tumor cell lines exhibited a “high-grade resistance” pattern. Detailed investigation of the SCD prodrug system suggested that the cytotoxic effect of converted 5-FU is directed either against the cells or against the virus particles, depending on the balance between cell line-specific susceptibility to GLV-1h94-induced oncolysis and 5-FU sensitivity. The data provided by this work underline that cellular resistance against VACV-based virotherapy can be overcome by virus-encoded prodrug systems. Phase I/II clinical trials are recommended to further elucidate the enormous potential of this combination therapy.Genetically modified vaccinia viruses (VACVs) have been shown to possess profound oncolytic capabilities. However, tumor cell resistance to VACVs may endanger broad clinical success. Using cell mass assays, viral replication studies, and fluorescence microscopy, we investigated primary resistance phenomena of cell lines of the NCI-60 tumor cell panel to GLV-1h94, a derivative of the  Cancer therapeutic resistance occurs as cancers develop resistance to treatments, such as chemotherapy, radiotherapy, and targeted therapies, through many different mechanisms. These include specific genetic and epigenetic changes in the cancer cell and/or the microenvironment in which the cancer cell resides. Beyond this, current standard therapies are also often burdened with a large number of serious side effects and organ damage. The main reason for this is the inability of these regimes to distinguish healthy tissue from tumor tissue. In contrast, novel biological therapeutics, such as oncolytic viruses, have exactly this capability to exert tumoricidal effects while sparing normal cells/tissues, e.g., by employing a unique primary oncolytic and secondary immunoinductive mechanism of action.Oncolytic viruses (OVs) mediate their anti-tumoral effect both via a direct and an indirect mechanism of action. They selectively infect and replicate in tumor cells. This massive replication finally leads to a metabolic breakdown of the cell and subsequently to cell lysis, resulting in the release of progeny virions which then can infect hitherto uninfected neighboring cells. In addition, OV-mediated cell death releases cytokines, tumor-associated antigens (TAAs), damage-associated molecular pattern molecules (DAMPs), and pathogen-associated molecular pattern molecules (PAMPs). These are taken up by antigen-presenting cells (APCs); in this course, CD4+ and CD8+ T cells are primed by cross-presentation. This activation of the adaptive immune system then also leads to an anti-tumoral immune response at tumor sites which have not been treated with virus ,2.Ankara (MVA) containing the yeast-originated transgene fcu1 (MVA-FCU1), expressing cytosine deaminase and uracil phosphoribosyltransferase enzymes, also called super cytosine deaminase (SCD), that transform the prodrug 5-fluorocytosine (5-FC) into cytotoxic 5-fluorouracil (5-FU) and 5-fluorouridine-5′-monophosphate, respectively [A variety of different virus strains , reovirus (REO), newcastle disease virus (NDV), herpes simplex virus (HSV), measles vaccine virus (MeV), and vaccinia virus (VACV)) are currently under intensive investigation not only in preclinical but also in clinical studies . Meanwhiectively . In a fiectively .Copenhagen strain) expressing the suicide gene fcu1 (VV-FCU1) can decrease the level of progeny virus particles. However, this inhibition of virus replication by 5-FC has no negative impact on the anti-tumor efficacy in diverse tumor cell lines as well as in a subcutaneous colon cancer mouse model [Numerous preclinical and clinical studies have already confirmed the efficacy and safety of viral therapeutics with integrated cytosine deaminase-based prodrug-converting systems ,11,12,13Lister derivative, yielding a replicating and thus oncolytic strain of vaccinia virus (GLV-1h94). Our rationale was to characterize GLV-1h94 for the treatment of solid tumors, which is why we investigated GLV-1h94 as monotherapy as well as in combination with 5-FC in 54 cell lines of the NCI-60 cell panel representing solid tumors. The main focus addresses the function and efficacy of virus-encoded suicide protein SCD. Moreover, primary resistance phenomena against virotherapy alone and the possibility to overcome this resistance with additional tumor-restricted/local chemotherapy were investigated.Encouraged by the promising data of the first-in-human study with MVA-FCU1, the prodrug-converting system SCD was transferred into a vaccinia Briefly, we could show that solid NCI-60 tumor cell lines responded with different levels of cellular resistance to GLV-1h94-based virotherapy. However, this resistance can be overcome by using the virus-encoded SCD prodrug system. Detailed investigation of the prodrug system revealed that the cytotoxic effect of converted 5-FU is directed either against the cells or against the virus particles, depending on the balance between cell line-specific susceptibility to GLV-1h94-induced oncolysis and 5-FU sensitivity.fcu1 suicide gene-encoding virotherapeutic vector GLV-1h94 alone  was assessed in a comprehensive and enlarged setting in 54 adherent cell lines derived from solid tumors of the NCI-60 panel, a well-established cancer cell line panel [First, the oncolytic efficacy of the ne panel . Due to All 54 tumor cell lines were infected with GLV-1h94 at MOI 0.1 and tumor cell masses remaining at 96 h post infection (hpi) were determined by a sulforhodamine B (SRB) assay. With regard to the results, three arbitrary response categories were defined a. Tumor ®) for immunotherapy of patients with unresectable, locally advanced or distant metastatic melanoma [As a result, more than half of the tested tumor cell lines (54%) turned out to be “high-grade permissive” regarding GLV-1h94-mediated oncolysis. Another 22% of the tested tumor cell lines were considered as “partially permissive” and 24% were found to be “high-grade resistant” a. In eacmelanoma . This dafcu1 suicide gene function , with active yeast cytosine deaminase (CD) expression and thymidine kinase (TK) deficiency, was evaluated in different cancer cell lines. Wild-type VG9 and VG9-CD both showed an identical oncolytic potential, which, however, was significantly different depending on the treated cell line. Their experiments also showed that the addition of 5-FC to VG9-CD-infected cells increased the oncolytic effect significantly compared to VG9-CD treatments alone. The authors postulate that the synergetic effect was more effective when the VG9-CD titers reached at least a level of MOI 1, indicating that the conversion efficiency of CD is only effective above a certain concentration of the expressed CD protein [Ankara (MVA) containing the yeast-originated transgene fcu1 (TG4023) was investigated in patients with metastatic liver tumors [8 pfu. Therapeutic 5-FU concentrations could be documented in tumors, indicating the proof of concept of virus-directed prodrug therapy. Furthermore, locally measured 5-FU concentrations were higher in tumor tissues compared to blood 5-FU levels, which might explain the reduced systemic toxicity of this special kind of tumor-restricted chemotherapy [Next, we investigated whether addition of the prodrug 5-fluorocytosine (5-FC) was able to enhance the anti-tumoral efficacy of GLV-1h94, thus exploiting the  protein . In a fir tumors . The cliIn this study, two of 29 tumor cell lines, which before were classified as “high-grade permissive” to GLV-1h94 monotherapy, now exhibited a “partially permissive” pattern when undergoing combined treatment (see breast cancer cell line BT-549 as well as lung cancer cell line NCI-H226). This phenomenon, on the one hand, might be explained by simple assay variations; on the other hand, it also might be reasonable to believe that converted 5-FU reduces the efficacy of the DNA virus GLV-1h94 due to its function as a thymidylate synthase inhibitor and thus not only prohibits genomic DNA but also viral DNA replication and consequently progeny virus synthesis.Based on these results, all following experiments were performed exemplarily only with the two “high-grade resistant” tumor cell lines NCI-H460 and HCT-15 as well as with the two “high-grade permissive” cell lines OVCAR-8 and DU-145.Based on the results of the NCI-60 tumor cell screening, which found that all tested cell lines reacted to the addition of 5-FC, but individually to varying extents, the 5-FU sensitivity of selected cell lines was investigated. For this assay, the two “high-grade resistant” tumor cell lines NCI-H460 and HCT-15, which showed significant tumor cell reduction with the addition of 5-FC after infection with GLV-1h94 b, and th−3 mM) were sufficient to reduce tumor cells to a remaining cell mass of ~60 % after 48 h and ~30 % , it was shown that tumor regression of B16 melanomas was not associated with progressive rounds of virus replication and subsequent oncolysis but rather correlated with viral gene expression and the induction of pro-inflammatory reactions in this tumor model .When investigating GLV-1h94 replication after prodrug activation by the addition of 5-FC, it became noticeable that in all tested cell lines, the formation of progeny virus particles was massively impaired compared to GLV-1h94 replication alone over the entire observation period of 96 hpi a,b. AccoTo further elucidate the impact of the chemotherapeutic compound 5-FU, the metabolic conversion rate of 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU) after infection of the “high-grade resistant” cell line NCI-H460 and the “high-grade permissive” cell line OVCAR-8 with GLV-1h94 and the addition of 5-FC was analyzed by mass spectrometry . Here, wAs expected, the conversion rates in both cell lines differed greatly from each other. Accordingly, the conversion rate in NCI-H460 cells at 96 hpi was below 50% a, whereaThese complex correlations indicate that the chemotoxic effect of converted 5-FU is directed against the tumor cells as well as against the virus particles. The balance between cell line-specific susceptibility to GLV-1h94-induced oncolysis and 5-FU sensitivity determines which effect predominates.To confirm and, more importantly, visualize the developed hypotheses, fluorescence and brightfield images of NCI-H460 and OVCAR-8  as well When observing the “high-grade permissive” cell lines OVCAR-8  tends to inhibit cell proliferation and thus indirectly virus replication, as the breeding ground for the production of progeny virus particles is missing.® (CCL-70TM) . All cell lines were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal calf serum . Cells were cultivated at 37 °C in a humidified atmosphere containing 5% CO2. Mycoplasma testing of all cell lines was performed regularly every three months .The US National Cancer Institute’s NCI-60 tumor cell panel was purchased from Charles River Laboratories . African green monkey kidney fibroblasts (CV-1 cells) were purchased from ATCCLister strain of vaccinia and were generated by homologous recombination of foreign genes into target loci of the VACV genome through double reciprocal crossover. In GLV-1h94, the gene locus for F14.5L was deleted by insertion of the fusion protein consisting of Renilla luciferase and Aequorea green fluorescent protein (ruc-GFP) under control of a vaccinia synthetic early/late promoter (PSEL). Furthermore, the gene locus for thymidine kinase (J2R) was deleted by insertion of ß-galactosidase ) and genes for human transferrin receptor , which allows for the detection of virus-infected cells at the protein level. Finally, the gene cassette A56R (encoding for hemagglutinin), which was deleted by insertion of ß-glucuronidase (gusA) in GLV-1h68, was replaced by the suicide gene fcu1 under control of PSEL. The expression of the converting enzyme SCD enables the transformation of 5-FC into 5-FU, which leads to RNA miscoding and direct inhibition of DNA synthesis.In this study, the vaccinia virus construct GLV-1h94, which was kindly provided by Genelux Corporation, San Diego, CA, USA, was used. GLV-1h94 b is a deGLV-1h94 was propagated in CV-1 cells. Cells were infected with GLV-1h94 at MOI 0.05. At 48 hpi, cells were harvested and homogenized in a Dounce homogenizer. Nuclei were removed by low-speed centrifugation. Virus containing supernatant was sonicated three times and then centrifuged through a sucrose cushion . Supernatant was discarded. The viral pellet was resuspended in Tris-HCl pH 9.0  and then sonicated again three times. Subsequently, virus was centrifuged through a continuous sucrose gradient (24–40%). The virus band was carefully harvested. The virus was centrifuged again and the pellet was resuspended in Tris-HCl pH 9.0. After three more sonication steps, the virus was stored in aliquots at −80 °C. Virus quantification was performed by titration on CV-1 cells.Respective cell lines were seeded in 24-well plates and infected with GLV-1h94 at a multiplicity of infection of 0.1 (MOI 0.1). At 3 h post infection (3 hpi), the inoculum was removed and growth medium with or without 1 mmol/l of the prodrug 5-FC  was added to the cells. At 96 hpi, the medium was removed and SRB assay was performed. As described previously , cells wTo investigate the replication of GLV-1h94, the respective cell lines were seeded in 6-well plates and infected with an MOI of 0.1. Cells were harvested by scraping them into their medium at 3, 24, 48, 72, and 96 hpi. Three subsequent freeze/thaw cycles led to cell lysis and to the release of cell-bound viral particles. To assess the count of viral particles in the harvested suspensions, serial dilutions of the samples were titrated on CV-1 cells in 24-well plates as described previously . After pCells were seeded in 6-well plates and infected with GLV-1h94 at an MOI of 0.01. At 3 hpi, the inoculum was removed and medium containing 0.1 mM 5-FC was added. Analyses were performed in triplicate. Supernatants were collected at 3 hpi, immediately after adding 5-FC, and at 24, 48, 72, and 96 hpi and incubated at 60 °C in a water bath for 30 min to inactivate the virus. Then, samples were snap-frozen in liquid nitrogen. For standard curves, cells were infected as described above. After inactivation of the virus, 5-FC and 5-FU were added to the supernatants at concentrations of 0, 0.01, 0.02, 0.04, 0.07, and 0.1 mM. For analysis, 100 µL methanol containing internal standard chlorouracil  were added to 400 µL sample or standard. Protein precipitation was performed by adding 800 µL acetonitrile. After 5 min of centrifugation at 13 000 g, 500 µL of the supernatants were evaporated at 60 °C in a vacuum evaporator and redissolved in 50 µL of 25% MeOH. Targeted quantification was achieved by injecting 2 µl sample extract into a 6500 QTRAP  mass spectrometer coupled to an Eksigent 200 microLC chromatography system . The autosampler was kept at 10 °C. For chromatographic separation, a BEH C18 column  at 50 °C column temperature and a gradient of two mobile phases  containing 0.2% formic acid; B: acetonitrile  containing 0.2% formic acid) at a 30 µL/min flow rate were used. The percentage of solvent B was raised from initially 2% for the first 0.2 min to 10% in 2 min and to 80% solvent B in another minute. After column cleaning with 98% solvent B, the solvent composition was returned to the initial start conditions. The mass spectrometer was run in multiple reaction monitoring (MRM) mode using positive ion mode electrospray ionization (ESI). Source settings were set to 5 kV as ion spray voltage, 150 °C as source temperature, and a curtain gas of 30. Mass transitions (m/z) and compound-specific settings can be found in Countless efforts aim to improve and optimize the treatment of cancer. In the early 19th century, observations were made that in many individual cases, tumor regression occurred with concurrent viral infections ,28. Thisfcu1 gene [In this study, the potential of recombinant vaccinia virus GLV-1h94 was examined, which was designed to express SCD, a prodrug-converting enzyme encoded by the cu1 gene . It coul


Tumor progression and tumor response to anticancer therapies may be affected by activation of oncogenic pathways such as the antioxidant one induced by NRF2 (nuclear factor erythroid 2-related factor 2) transcription factor and the pathways modified by deregulation of oncosuppressor p53. Often, oncogenic pathways may crosstalk between them increasing tumor progression and resistance to anticancer therapies. Therefore, understanding that interplay is critical to improve cancer cell response to therapies. In this study we aimed at evaluating NRF2 and p53 in several cancer cell lines carrying different endogenous p53 status, using a novel curcumin compound since curcumin has been shown to target both NRF2 and p53 and have anti-tumor activity.We performed biochemical and molecular studies by using pharmacologic of genetic inhibition of NRF2 to evaluate the effect of curcumin compound in cancer cell lines of different tumor types bearing wild-type (wt) p53, mutant (mut) p53 or p53 null status.We found that the curcumin compound induced a certain degree of cell death in all tested cancer cell lines, independently of the p53 status. At molecular level, the curcumin compound induced NRF2 activation, mutp53 degradation and/or wtp53 activation. Pharmacologic or genetic NRF2 inhibition further increased the curcumin-induced cell death in both mutp53- and wtp53-carrying cancer cell lines while it did not increase cell death in p53 null cells, suggesting a cytoprotective role for NRF2 and a critical role for functional p53 to achieve an efficient cancer cell response to therapy.These findings underline the prosurvival role of curcumin-induced NRF2 expression in cancer cells even when cells underwent mutp53 downregulation and/or wtp53 activation. Thus, NRF2 inhibition increased cell demise particularly in cancer cells carrying p53 either wild-type or mutant suggesting that p53 is crucial for efficient cancer cell death. These results may represent a paradigm for better understanding the cancer cell response to therapies in order to design more efficient combined anticancer therapies targeting both NRF2 and p53. The oncosuppressor p53 plays a key role in cell growth and apoptosis in response to various stress signals . Given iNRF2 is the main regulator of cellular antioxidant response  and is aCurcumin is considered a chemopreventive molecule with anti-oxidant, anti-apoptotic and anti-inflammatory properties and with an excellent safety profile although it presents low solubility and bioavalibility . CurcumiIn this study we aimed at evaluating the effect of a novel curcumin compound in several cancer cell lines carrying different endogenous p53 status. We used a water-soluble ruthenium(II)-curcumin (RuCUR) compound that presents high solubility and cytotoxic effect ]compared to other similar curcumin compounds –46. MoreThe water-soluble ruthenium(II)-curcumin compound (RuCUR) was synthesized as described previously , 43. In The cell lines used in this study were: human SKBR3 , T98MG , MCF7 breast cancer, U87 glioblastoma, HCT116 and RKO colon cancer cell lines , and HCT116 p53 null. The cells were cultured in either DMEM (Dulbecco modified Eagle’s medium) (Life Technologies-Invitrogen), or RPM1–1640 (Life Technologies-Invitrogen), with 10% heat-inactivated foetal bovine serum (FBS)  and L-glutamine/streptomycin (100 μg/mL) , in 5% CO2 at 37 °C. They were all mycoplasma negative. The NRF2 inhibitor Brusatol   staining and direct counting with a Neubauer haemocytometer, as previously reported .The 3D spheroids, were obtained into ultra-low attachment cell culture multiwell plates , as previously reported , 50. WheReactive oxygen species (ROS) production was assessed by using the 2′,7′-dichlorofluorescein diacetate  staining, as previously reported . Cell peCell pellets were lysed in lysis buffer   and a mix of protease inhibitors  and protein concentration determined with the BCA Protein Assay  to load equal amount of total cell extracts on 9–18% SDS-polyacrilamide gels (PAGE)  electrotrophoreses. After blotting to polyvinylidene difluoride membranes . Unspecific sites were blocked with 5% non-fat powdered milk or with 3% bovine serum albumin  in 0.05% Tween-20 (v/v in TBS). Membranes were then incubated with primary antibodies. The antibodies we used in this study were: mouse monoclonal anti-p53 (DO-1) (1:1000) (sc-126), rabbit polyclonal anti-p21 (C-19) (1:500) (sc-397), mouse monoclonal anti-HO-1 (A-3) (1:1000) , mouse monoclonal anti-p62/SQSTM1 (D-3) (1:1000) , all from Santa Cruz Biotechnology; rabbit polyclonal anti-LC3B (1:2000) ; mouse monoclonal anti-NQO1 (A180) , mouse monoclonal anti-HSP90 (1:1000) , rabbit polyclonal anti-NRF2 (1:1000) , mouse monoclonal anti-poly(ADP-ribose) polymerase (1:1000) , mouse monoclonal anti-phospho-Histone H2AX (1:1000)  , and rabbit polyclonal anti phospho-4E-BP1 (236B4) . Mouse monoclonal anti-actin antibody (Ab-1) (JLA20) (1:10.000)  was used as loading control. Anti-imunoglobulin-G-horseradish peroxidise (HRP) secondary antibodies that we used were: anti-mouse IgG-HRP  and anti-rabbit IgG-HRP . Enzymatic signals were visualized by chemiluminescence , according to the manufacturer’s protocol.Total RNA extraction was performed by using TRIzol Reagent (Life Technology-Invitrogen), cDNA was synthesized by using MuLV reverse transcriptase kit  and semi-quantitative Reverse-Transcribed (RT)-PCR was carried out by using Hot-Master Taq polymerase , as previously reported . DensitoFor RNA interference, cells were transfected with the Nrf2 siRNA  and control siRNA  using LipofectaminePLus reagent , following the manufacturer’s instructions, as previously reported .http://imagej.nih.gov) and relative band intensity normalized to β-actin signals.Quantification of the protein bands was assessed by densitometric analysis using the ImageJ software  of at least three independent experiments. Two-tailed Student’s t-test and one-way ANOVA analysis were used for statistical significance of, respectively, two o more sample comparisons. Difference was considered statistical significant when We first evaluated the effect of RuCUR compound on cancer cell lines carrying mutp53 by performing a three-dimensional (3D) culture spheroids proliferation assay , 50. CelNext we evaluated the effect of RuCUR compound on cancer cell lines carrying wtp53. We found that increasing doses of RuCUR compound significantly inhibited spheroids proliferation -curcumin compound (RuCUR)  was ableMutp53 proteins may promote tumor invasion, metastasis and chemoresistance, disclosing mutp53 as a guardian of the cancer cells . RestoraCip1/WAF1 levels [Curcumin may induce NRF2 pathway activation . NRF2 in1 levels . We foun1 levels , 52. The1 levels –75, alth1 levels , in a coThis study suggests that the overexpression of the antioxidant system is involved in the mechanism of cancer cell resistance to RuCUR treatment. The elimination of mutp53 and the activation of wtp53 induced cell death although only after inhibition of the antioxidant system cell death greatly improved. Interestingly, p53 null cells did not undergo increased cell death following inhibition of the NRF2 pathway, underscoring the important role of p53 in cancer cell death. These findings also suggest that the use of phitochemicals that can increase the antioxidant system and, for this reason, being useful antiaging agents, may not be beneficial for the demise of tumors, despite autophagy induction targeting oncogenes such as mutp53, underlining the complex relationship between autophagy, antioxidant system and tumor cell death. In conclusion, these findings may represent a paradigm for better understanding the complex interplay between NRF2 and p53 molecular pathways, in order to design more efficient anticancer therapies. Further preclinical and clinical investigations of RuCUR /NRF2 inhibition combination should be performed in order to explore the anticancer activities.


Although worldwide real and pilot applications show promising results, the effects of nZVI on exposed living organisms are still not well explored. The majority of the recent studies examined toxicity to microbes and to a lesser extent to other organisms that could also be exposed to nZVI via nanoremediation applications. In this work, a novel approach using amoebocytes, the immune effector cells of the earthworm Eisenia andrei, was applied to study the toxicity mechanisms of nZVI. The toxicity of the dissolved iron released during exposure was studied to evaluate the effect of nZVI aging with regard to toxicity and to assess the true environmental risks. The impact of nZVI and associated iron ions was studied in vitro on the subcellular level using different toxicological approaches, such as short-term immunological responses and oxidative stress. The results revealed an increase in reactive oxygen species production following nZVI exposure, as well as a dose-dependent increase in lipid peroxidation. Programmed cell death (apoptosis) and necrosis were detected upon exposure to ferric and ferrous ions, although no lethal effects were observed at environmentally relevant nZVI concentrations. The decreased phagocytic activity further confirmed sublethal adverse effects, even after short-term exposure to ferric and ferrous iron. Detection of sublethal effects, including changes in oxidative stress-related markers such as reactive oxygen species and malondialdehyde production revealed that nZVI had minimal impacts on exposed earthworm cells. In comparison to other works, this study provides more details regarding the effects of the individual iron forms associated with nZVI aging and the cell toxicity effects on the specific earthworms’ immune cells that represent a suitable model for nanomaterial testing.During the last two decades, nanomaterials based on nanoscale zero-valent iron (nZVI) have ranked among the most utilized remediation technologies for soil and groundwater cleanup. The high reduction capacity of elemental iron (Fe After incubation with 1, 10 and 100 mg/L nZVI NPs after 2, 6 and 24 h, the beads were added at a ratio of 1:100 (cells:beads) and incubated for 18 h at 17 °C. After incubation, the cells were washed twice with PBS , stained with PI (1 mg/L) and analyzed by flow cytometry. Finally, the phagocytosis is expressed as a percentage of engulfed fluorescence beads by the cells.The phagocytic assay was performed with fluorescent beads , 0.14M NaCl, and 2.5 mM CaCl2 solution) and stained with 30 µL of Annexin V  for 15 min in the dark at room temperature. Prior to flow cytometry analysis, the suspended cells were stained with PI .Viability and cell death stage assays were performed after incubation of the coelomocytes with 1, 10 and 100 mg/L nZVI NPs. The cell suspension was washed twice with Annexin V buffer  were used as positive controls for the endpoints measured by flow cytometry. The forward and side scatter and stain fluorescence settings were adjusted for each coelomocyte subtype and fluorescent probe  by GraphPad Prism Cellular ROS production, viability, phagocytic activity, and the stages of cell death were analyzed with a flow cytometer . To assess the reliability of these assays, 10 mM and 100 mM Hnt probe . For ana2 NPs conducted by Márquez et al. (2017) [The results of SEM examination revealed an increase in aggregate formation between fresh nZVI NPs and nZVI NPs aged for 24 h in distilled water a,b. Howe. (2017) .2 (source of Fe2+ ions) and Fe2(SO4)3 (source of Fe3+ ions). Notably, under specific conditions, nZVI can be oxidized/transformed into ferric and ferrous species that can be involved in ROS generation through Fenton-like processes [Moreover, ICP-OES analysis of dissolved iron demonstrated that ions were released from the nanomaterial . The disrocesses ,37.Viability analysis did not show any significant effect following exposure of both granular and hyaline amoebocytes to either nZVI NPs at the tested concentrations or the tested ionic forms of iron . The absIn addition to viability studies, the use of flow cytometry allowed the monitoring of cell death stages, including apoptosis and necrosis, in coelomocytes. Moreover, based on the combination of different staining (PI and Annexin V) for the detection of membrane integrity and lipid phosphatidyl serine externalization , the early and late apoptosis were determined in the exposed cells. Only the ferric form of iron significantly decreased early apoptosis of granular amoebocytes (after 6 and 24 h) and increased their necrosis after 6 h . In cont2+ and Fe3+ for 2 h and in granular amoebocytes exposed to Fe3+ for the same amount of time. The differences in ROS production between GA and HA caused by Fe2+ and Fe3+ could be explained by different sensitivities of the two cell types towards various redox forms of iron ions. Different antioxidative defense, uptake and biokinetics could result in different ROS production induced by Fe2+ and Fe3+. Interestingly, after longer exposure (6 or 24 h), the production of ROS species decreased and did not differ significantly from that in the untreated controls. Several authors have found downward trends in ROS generation following longer exposure periods, and one possible explanation for this phenomenon is the activation of the antioxidative defense system [Despite the fact that Yirsaw et al. (2016) did not observe a decrease in the survival of earthworms exposed to nZVI NPs, they were able to detect various concentration-dependent sublethal effects, such as lipid peroxidation and DNA damage . In the e system ,40. Celle system ,41. The e system . These r2+ and Fe3+) also induced a significant increase in lipid peroxidation compared to that in the control sample. The same concentration of iron in the form of ferrous ions (Fe2+ dissolved from FeCl2) produced significantly more MDA than iron in the form of ferric ions (Fe3+ dissolved from Fe2(SO4)3). Determination of MDA levels was the most sensitive assay out of those used, as in a previous study focused on adverse effects in bacteria [Analysis of lipid peroxidation in coelomocytes after exposure to nZVI NPs showed a dose-dependent increase in MDA production . Even thbacteria . The appbacteria ,43. The bacteria .The phagocytic activity of hyaline amoebocytes was not affected by the tested concentrations of nZVI NPs or iron ions . The graEscherichia coli exposed to nZVI NPs than to Escherichia coli exposed to the same concentration of ferrous iron under both air-saturated and deaerated conditions [2 and Fe2(SO4)3 that reached from 50 to 100 times higher concentrations  and ferrous and ferric ions, as potentially released forms of iron, do not significantly affect the viability of coelomocytes and probably do not pose a risk to exposed earthworms. Determination of the sublethal effects including impacts on phagocytosis and oxidative stress-related markers showed that ferric and ferrous ions as well as nZVI NPs aggregates had a slight effect. The combination of the quantification of dissolved iron and the most sensitive assay (from the battery of assay used), malondialdehyde determination, showed that, even in the form of aggregates, nZVI NPs cause higher lipid peroxidation than their potential transformation products  in earthworm coelomocytes. Overall, the results of the present study confirm the environmental safety of nZVI NPs and suggest that the toxicity of nZVI NPs is potentially reduced during aging. Further studies incorporating the nZVI NPs characterization during the aging process and iron dissolution under different environmental conditions are needed to better understand material behavior and toxicity in real-world nanoremediation applications. Our results also show that the methodological concept of testing isolated immune earthworm cells is a suitable approach to testing for nanomaterials toxicity and to elucidate mechanistical aspects of the nanomaterials with respect to possible different modes of actions.