An incubation experiment was conducted to evaluate the effect of nanoparticles of zero-valent iron (nZVI) on Ni+2 sorption in biosolids-treated agricultural soils. Two application rates of biosolids  and four treatment levels  of nZVI were examined, either separately or interactively. The results of this study showed significant differences in Ni+2 sorption capacity between different nZVI treatments. The initial Ni+2 concentration in biosolids-amended soil significantly affected Ni sorption in the soil treated with nZVI. The “H-shape” of sorption isotherm in nZVI-treated soil reflects strong interaction between the Ni concentration and the nZVI treatment, while the C-shape of sorption isotherm in biosolids-amended soil without the nZVI treatment indicates intermediate affinity for Ni+2 sorption. Nickel retention in soil was increased with the increase of nZVI levels. The removal efficiency of Ni+2 by nZVI from solution was increased with the increase of pH from 5 to 11 and reached a maximum of 99.56% at pH 11 and nZVI treatment of 10 g/kg. The Ni+2 desorption rate decreased from 92 to 7, 4, and 1% with increasing nZVI treatment levels from 0 to 1, 5, and 10 g/kg, respectively, with a soil Ni+2 concentration of 50 mg/L. The maximum adsorption capacity (𝑞max) of 10 g/kg nZVI-treated soil was 333.3 mg/g, which was much higher than those from the other treatments of 0 (5 mg/g), 1 (25 mg/g), and 5 g/kg (125 mg/g). The underlying mechanism for Ni+2 immobilization using nZVI in an aquatic environment is controlled by a sorption process, reduction of metal ion to zero-valent metal, as well as (co)precipitation. Moreover, increasing the nZVI treatment level in biosolids-amended soil significantly decreased bioavailable Ni+2 concentrations in the soil.Nickel (Ni Thus, the land application of biosolids may result in toxic Ni+2 contamination in biosolids-amended agricultural soil and also in land surface runoff.Soil contamination with heavy metals, such as nickel, is a significant and global problem . Anthrop+2-contaminated soil and water can be achieved through different physical, chemical, and biological processes. For example, Ni2+ can be removed from contaminated soils and waters by chemical adsorption and precipitation using nanoscale zero-valent iron (nZVI) [2+ to Ni0 will also reduce bioavailability of Ni+2 in contaminated soils [+2-contaminated biosolids.In situ remediation of Nin (nZVI) ,4,5. Cheed soils ,7. Nanosed soils ,9,10 haved soils ,12, incled soils ,14,15. W+2 in soil and water, and (3) determine the possible changes in Ni fractions in the biosolids-amended soil.The specific objectives of this study were to (1) examine the factors that influence the Ni sorption on nZVI in biosolids-amended agricultural soils, (2) evaluate the retention and removal efficiencies of nZVI for NiTypic xeropsamments) from El-Bostan, El-Behaira governorate, Egypt was selected for this incubation experiment. Surface soils (0–30 cm) were sampled, air-dried, ground, and sieved through 2 mm. Biosolids  were collected from the Municipal Wastewater Treatment Plant in Alexandria (Station No.26), Egypt, and air-dried, ground, and sieved (<2 mm) [A sandy loam soil ( (<2 mm) . The nan (<2 mm)  and compw/w) and four nZVI treatment levels of 0, 1, 5, and 10 g/kg, either separately or combined. Concentration of Ni in the collected biosolids was 6.23 mg Ni/kg. The soil mixes were put in different jars, moistened, and equilibrated at 80% of the water holding capacity at 25 ± 2 °C for 6 days. This incubation experiment was carried out in a CRD design with three replicates. At the end of the incubation period, the soil sampling was carried out and prepared for future analyses.A laboratory incubation experiment was conducted to stabilize the experimental soils being treated with biosolids and/or nZVI for equilibrium, including two biosolids application rates of 0 and 5%  at equilibrium; qe is the sorption capacity (mg/g) at equilibrium; qmax is the maximum sorption capacity (mg/g); KL is the sorption constant of Langmuir (L/mg) at equilibrium; KF and n are Freundlich isotherm constants related to the sorption capacity.On the contrary, the Freundlich sorption isotherm model can describe the Niessed as :qe = KFC+2 treatment level of 800 mg/L and to quantify adsorption isotherms for Ni+2 in control soil and the soils amended with biosolids and treated with nZVI particles at 25 °C at different time points of 5, 30, 60, 120, 240, 480, 960, and 1440 min.The batch method described by Amacher et al.  was used+2, two common (first-order and power function) kinetic models were applied to the sorption data. The two equations are as follows [To understand the effect of shaking extraction time on the sorption process of Ni follows ,23:Firstq = the amount of sorbed Ni per gram of soil mix in a specific time ta = the coefficient of sorption diffusion rate (μg/ g min)Ko = the initial Ni+2 concentration (mg/ L)Ct = reaction time (min)a = the coefficient of sorption rate (min−1)k1/m = a constanto = the amount of sorbed Ni at equilibriumqFractions of nickel in the soil amended with biosolids was conducted for both nZVI-treated and un-treated samples utilizing the successive extraction procedure according to Tessier 1979 . The ext+2-loaded nZVI was studied to test its stability. The amount of de-ionized water (10.0 mL) was added to 1.0 g Ni-loaded nZVI and shaken for 24 hrs. After shaking and centrifugation, the suspension solutions were then filtered. The Ni concentration was measured in filtrates by ICP.Desorption behavior of Ni+2 from contaminated water was studied using packed-column. The column was made of clear acrylic  and filled with 20 g coarse sand; 1, 5, or 10 g nZVI treated and biosolid-amended soil; and 10 g fine sand. A total of ten columns, including two control columns (control and biosolids-amended soil without nZVI treatment), were constructed to evaluate the performance of the columns at four doses:sand ratios. Industrial wastewater effluents (100 mL) spiked with Ni+2 of 56.23 mg/L was continuously pumped into the columns, and effluent concentrations were measured with performance time. The performance of the packed bed column under different conditions was evaluated from breakthrough curves, and the optimal application rate of nZVI for best pollutant stabilization was determined.Under continuous flow conditions, the potential of nZVI to remove Ni+2 concentrations were determined.The five pore volumes of the injection solution were then pumped into the column in a downward direction at 0.5 ml/min flow rate. Effluent samples were collected, and Ni+2 removal column for nickel sorption (q) was calculated:+2 sorbed per gram of nZVI (mg/g), Co is the initial concentration of Ni (mg/L), Vo is the total volume of the influent solution (L), Cn is the concentration of Ni in sample n (mg/L), Vn is the volume of sample n (L), and m is the amount of nZVI (g). After finishing the experiment, the nickel-loaded nZVI will be regenerated and can be reused for another number of cycles [The capacity of the Nif cycles ,25.2O3) and magnetite (Fe3O4), respectively.The X-ray diffraction (XRD) patterns of nZVI showed apparent peaks 2θ = 45° and 64.88°, indicating the presence of metallic iron products . The chaThe morphology of nZVI particles was analyzed by scanning electron microscopy (SEM) detection, and the results clearly showed that the representative single particle size dimension lies in the range 1–100 nm (nanostructure), the nZVI particles are spherical with an average size of 42–45 nm, and the particles are uniformly distributed. The energy dispersive Xray (EDX) elemental analysis of nZVI confirmed the presence of Fe in the sample by appearance of Fe (56.54%) peak in nZVI sample. Moreover, the size distribution of Zetasizer analysis refers to size average 43 nm (<100 nm).2/g) was quite high. Indeed, the high SSA could supply nZVI with highly reactive sites for Ni+2 sorption. These results coincided with the results of Mahdy et al. [The specific surface area (SSA) of nZVI  A.It was speculated that the nZVI treatments could contribute many more sorption sites for ZVI nanoparticles, as well as iron hydroxides in the soils. The high specific surface area of nZVI and amorphous iron hydroxides and additional negative charges from biosolids amendment could significantly increase the cation exchange capacity (CEC) in the soil .+2 to Ni occurred due to a slightly higher standard potential of Ni than that of Fe. During the reduction process, metallic iron will serve as an electron donor and provide electrons to reduce Ni (II) [At a solution containing Ni concentration of 800 mg /L, the amount of sorbed Ni in 5% biosolids-amended soil was 7500, 73,245, 75,022, and 76,888 mg Ni/kg when the soil was treated with nZVI of 0, 1, 5, and 10 g/kg soil, respectively A. High s Ni (II) ,29.Fe0 Increasing the nZVI treatment level from 1g/kg to 10 g/kg would also increase the efficiency of Ni retention in the soil B.+2 concentration of 50 mg/L, the Ni+2 removal efficiency from solution increased from 71.3% to a maximum 96.2% when the nZVI treatment level increased from 1 to 10 g/kg. However, at 800 mg Ni+2/L, the efficiency increased from 91.6% to 96.1% with the increase of nZVI treatment level from 1 to 10 g/kg. Further, a similar efficiency was also observed at initial Ni concentrations of 100, 200, and 400 mg/L . Its sorption behavior in aquatic environments has previously been reported ,38,39. I+2 retention in soil after addition of the nZVI to the biosolids-amended soil (data not shown). The FTIR spectra of the nZVI-biosolids-amended soil showed stretching and bending of HOH, as well as bending vibration of hydroxyl groups on metal oxides; disappearing and shifting of some peaks was noticed. These observations suggest that chemical reactions occurred between Ni+2 and OH groups on nZVI particle surfaces.The Fourier transmission infrared spectroscopy (FTIR) analysis was performed to investigate the mechanism of Ni+2 sorbed on biosolids-amended soils treated with nZVI at both 50 and 800 mg Ni/L  of 10 g/kg nZVI-treated soil was 125 mg/g, which was much higher than those from the other treatments of 0 (5 mg/g), 1 (25 mg/g), and 5 g/kg (33 mg/g). Indeed, the specific surface area and the nZVI treatment in biosolids-amended soil increases sorption sites on soil surface. High specific surface area of nanoparticles greatly enhanced the adsorption capacity and surface reactivity of nZVI-treated soil [As shown in ted soil .+2 sorption in the biosolids-amended soil treated with nZVI  was observed with the nZVI treatment of 10 g/kg but the lowest (3.69 mL/g) with 0 g/kg nZVI treatment. Clearly, increasing nZVI treatment levels added to biosolids-treated soil greatly increased its capability for Ni+2 adsorption or removal from aqueous solutions. The 1/n values of Freundlich isotherm were within a narrow range (1.03–1.71) for the three studied nZVI dosages, in addition to control treatment, and reflect the observed similarities of the overall shape of Ni isotherms, as shown in +2 sorption on soil–biosolids–nZVI mix at a concentration range of 50–800 mg Ni+2/L of solution. However, the SE values calculated using the Freundlich model for Ni+2 sorption in the biosolids- and nZVI-treated soil were much higher than those of the Langmuir model, which indicates the superiority of the Langmuir model over the Freundlich model in describing Ni+2 sorption in the soil treated with biosolids and nZVI.The Freundlich isotherm model was also used to describe Niith nZVI B. Nickelted soil . Table 22+ sorption equilibrium in the soil treated with biosolids and nZVI were assessed by performing kinetic experiments. The Ni+2 sorption or removal efficiency versus reaction time is shown in The sorption capability of nZVI as sorbent and determination of the time required for Ni+2 solutions, the maximum Ni+2 removed by nZVI/biosolids-treated soil were 73,245, 75,022, and 76,888 mg/kg at 1, 5, and 10 g nZVI/kg, respectively, as the equilibrium was reached after 24 hrs  by nZVI, first-order  and poweThe relatively high percentage of Nickel bound to Fe–Mn oxides in the soil was mainly due to application of nZVI to biosolids-treated soil. Moreover, the increase of residual fraction of the Ni form after addition of nZVI was due to the conversion of all available and moderately-available forms of Ni into unavailable forms of nickel as a residual fraction . The bioavailability of nickel in the 5 % biosolids-amended soil treated with different levels of nZVI followed the order of: control soil without nZVI treatment > 1 g/kg nZVI-treated soil > 5 g/kg nZVI-treated soil > 10 g/kg nZVI-treated soil . These r+2 by nZVI was studied. The flow rate of nickel-contaminated solution was 0.5 mL/min and the Ni+2 concentration in the solution was kept at 50 mg/L  by nZVI from solution was increased with increasing pH in the range of 5 to 11 and reached a maximum of 99.56% at pH 11 and nZVI treatment of 10 g/kg. The Ni desorption rate from soil–biosolids–nZVI mix loaded with Ni was decreased from 92 to 7, 4, and 1% with increasing nZVI content from 0 to 1, 5, and 10 g/kg at an initial Ni concentration of 50 mg/L. The maximum adsorption capacity (𝑞max) value of 10 g/kg nZVI-treated soil  was much higher than those of 0, 1, and 5 g/kg . The possible mechanism underlying nZVI-immobilization of heavy metal ions in an aqueous system is controlled by sorption, reduction of metal ion to zero-valent metal, and (co)precipitation. Moreover, increase of nZVI content to biosolids-amended soil significantly decreased Ni+2 concentrations and mobility in the packed-column in the five pore volumes. Therefore, it is suggested that the obtained nZVI could be used as a good sorbent for the removal of Ni+2 from wastewater. It appears that iron nanoparticles are effective for the immobilization of nickel present in biosolids -treated soil or Ni+2-contaminated aqueous solutions and potentially have advantages over conventional methods for remediation or stabilization of heavy metals in contaminated soils. More significantly, iron nanoparticles can effectively reduce and immobilize nickel in contaminated soil due to their extremely small particle size and penetration into the intra-aggregate pores of contaminated soil. Moreover, the end products of iron nanoparticle reactions are iron hydroxides and oxides, similar to the iron minerals ubiquitous in the natural environment. The addition of small amounts of iron nanoparticles may, in fact, enhance the nutritional value of biosolids for land applications. The potential of iron nanoparticles for the remediation of biosolids-contaminated soil merits further investigation.The results of this study revealed that there were big differences in Ni


Risedronate sodium is frequently used in the therapy of bone tissue disorders. Our study demonstrated increased efficiency of cytostatic drugs in the presence of risedronate. During the incubation, testing and evaluation the standard protocols were used. MTT and TUNEL assays were employed to estimate changes in cell viability and percentage of apoptosis. We found that a combination of doxorubicin and cisplatin with risedronate sodium produced the strongest cytotoxic effects in both cell cultures. The cytotoxicity against both types of osteosarcoma was concentration-dependent and the cytostatic drugs and risedronate sodium were found to act synergistically.The study investigated canine (D-17) and human (U-2 OS) osteosarcoma (D-17 and U-2 OS). Standard protocols were used for the preparation of cell cultures and evaluation of their viability and apoptosis. MTT assay assessed the culture viability and EC50, while the apoptotic effect of the drugs was checked with a TUNEL assay. Doxorubicin alone showed the strongest cytotoxicity against D-17 (0.056 ± 0.019 µg/mL) and U-2 OS (0.051 ± 0.003 µg/mL), while the lowest cytotoxicity was observed for carboplatin . Risedronate sodium at 100, 10 and 1 µg/mL lowered viability in OS cell lines by 53.38 ± 1.46 and 49.56 ± 0.7%, 97.08 ± 3.32 and 74.92 ± 4.01%, and 102.67 ± 3.56 and 94.56 ± 3.52%, respectively. In all analyzed drug combinations, risedronate sodium significantly (* p < 0.05) increased the cytotoxicity against tested osteosarcoma cell lines. The decrease in cell viability caused by the studied compound combinations was weaker in canine than in human cell cultures. A combination of doxorubicin , cisplatin (1 µg/mL) and etoposide (1 µg/mL) with 100 µg/mL of risedronate sodium significantly improved the cytotoxicity of the drugs against canine and human osteosarcoma. Administration of carboplatin (1 µg/mL) and risedronate sodium (100 µg/mL), compared to carboplatin per se, produced no significant differences in cytotoxicity against the D-17 cell culture but significantly enhanced cytotoxicity in the U-2 OS line. The strongest apoptosis in both lines was detected for 0.01 µg/mL doxorubicin combined with 100 µg/mL risedronate sodium or 1 µg/mL cisplatin and 100 µg/mL risedronate sodium. In all combinations, the tested compounds revealed a synergistic mechanism of action.The study discusses in vitro cytotoxicity of a combination of cytostatic drugs  and risedronate sodium against canine and human  Osteosarcoma (OS) is the most common primary bone tumor, diagnosed in 80–85% of canine patients with bone neoplasms. The disease is an important problem of human and veterinary oncology. This neoplasm is a primary bone tissue malignancy of mesenchymal origin and highly diverse histopathological structure. Considering all diagnosed neoplasms in dogs, the prevalence of OS is moderate. It occurs usually in adult large and giant breed dogs (2–15 years old) [ars old) ,4,5,6. Fars old) .The histopathology, location and predisposing factors of canine and human OS are highly similar . ChildreThe newest generation of bisphosphonates (risedronate sodium) have the strongest affinity for tissues of the skeletal system. Risedronate molecules accumulate in the bone matrix. They do not penetrate the cell cytoplasm but inhibit hydroxyapatite degradation and decrease osteoclast activity by inhibition of the mevalonate pathway leading to apoptosis . The accPlatinum derivatives chosen for this study (cisplatin and carboplatin) are inorganic, hydrophilic and DNA alkylating compounds. Cisplatin or carboplatin are cytostatics routinely used in humans and animals. The mechanism of their cytotoxic action consists of binding to numerous endogenous compounds with nucleophilic functional groups  that are typical not only for DNA but also for RNA, enzymes or hormones . CisplatCarboplatin is a derivative of cisplatin with similar pharmacological characteristics and a similar mechanism of action.Podophyllum sp. The drug is used in the medical treatment of humans and dogs. Its pharmacodynamics are not fully elucidated but etoposide affects the activity of topoisomerase II and subsequently causes DNA damage [Etoposide is a plant based semi-synthetic derivative of podophyllotoxin extracted from A damage . This maA damage . ResistaStreptomyces peucetius v. cesius. The drug is used in canine, feline, human and ferret anticancer therapy. Its mechanism of action is based on doxorubicin incorporation into the DNA helix and inhibition of topoisomerase II activity [Doxorubicin is a compound of natural origin isolated from activity . Additioosteosarcoma  or other neoplasms . Risedronate sodium combined with those agents may change the cytotoxic effects observed in the investigated cell cultures, either by synergy or addition.The objective of our study was to assess the pharmacological interaction between risedronate sodium and selected cytostatic drugs with cytotoxic effects against 2)  with OS cell lines  were incubated in standard conditions . Supplementation of an Eagle’s Minimum Essential Medium (EMEM) and a McCoy’s 5A culture medium  with 10% fetal bovine serum (FBS) , 4 nM L-glutamine , 100 U/mL penicillin, and 100 µg/mL streptomycin  was used.Culture flasks  was added to OS cell cultures to peel them off the flask bottom. The cell suspension (10 µL) was mixed with 10 µL of Trypan blue  and 80 µL of PBS . The number of cells was calculated by a hemocytometer.The drugs  were tested at the following concentrations: doxorubicin 1, 0.5, 0.1, 0.05, 0.01, 0.005 and 0.001 µg/mL; cisplatin and carboplatin 50, 20, 10, 5, 1, 0.5, 0.1 and 0.05 µg/mL; etoposide 10, 5, 2.5, 1, 0.5, 0.1, 0.05, 0.025, 0.01, 0.005 and 0.0025 µg/mL; and risedronate sodium 300, 150, 100, 30, 15, 10, 3, 1.5, 1, 0.3 and 0.15 µg/mL.3/100 µL per well, under the same conditions as in the flasks. Then the medium (EMEM and McCoy’s 5A) was changed and the cultures were exposed to the studied compounds alone (as mentioned above) or in combinations  at a density of 3 × 10inations  for 72 hAfter 24 h incubation the absorbance was measured with a spectrophotometer . The viability of cells was estimated according to the following formula :Viabilit50 were computed. For drug combinations only the mean value was calculated.Four independent repetitions were carried out for the individual substances and the mean value and EC50), were chosen for further tests. This procedure allowed for confirmation of pharmacological interactions between the tested substances. The results underwent a statistical analysis. The combinations of drug concentrations selected for further analysis are shown in The concentrations that decreased the cell viability by more than 50% .The share of apoptotic cells was estimated using the ApopTag4 cells/40 µL of the medium were placed in 10-well polytetrafluoroethylene coated slides  and incubated for 24 h. Then, the tested compounds were added after the medium removal (2) and the cells were incubated for about one minute. Directly after the buffer removal, the cell cultures with 55 µL/5 cm2 of Working Strength TdT Enzyme were incubated at 37°C, in a moist chamber, for 1 h. Next, the slides were put into Stop/Wash Buffer for 10 min and subsequently rinsed in PBS (3 × 1 min). At the next step, 65 μL/5 cm2 of Anti-digoxigenin Peroxidase Conjugate was added to each well and the slides were incubated in the moist chamber for 30 min at RT. After another cycle of rinsing in PBS (4 × 2 min), 75 μL/5 cm2 of DAB Peroxidase Substrate was added and the hydrophobic slides were incubated at RT for 10 min. Finally, the slides were washed in distilled water (3 × 1 min) and incubated for 5 min in bi-distilled water. The cell nuclei were stained with 1% haematoxylin  and dehydrated in 70% ethanol for 30 s and in xylene for 30 s . The final stage of the procedure involved mounting a cover glass on each slide using DPX .The tested cultures of OS cell lines at a concentration of 2 × 10 removal . After tThe light microscope Olympus BX53  was used to estimate the percentage of apoptotic cells and to count them . The results are presented as the mean for each field of vision . The prop = 0.05.The results were analyzed using the StatisticaPL 10.0 package . The normal distribution of values was tested using the Shapiro-Wilk test, and comparison of drug combinations was carried out using the Kruskal-Wallis analysis. The results on canine and human OS were compared with the Mann-Whitney U test. The Spearman’s correlation coefficients were employed. Statistical significance was assumed at 50 reached 0.056 ± 0.019 µg/mL and 0.051 ± 0.003 µg/mL, respectively. Of the tested drugs carboplatin showed the weakest cytotoxicity, with EC50 6.45 ± 0.2 µg/mL and 27.5 ± 2.3 µg/mL for the canine and human OS cell line, respectively. Only one form of the third generation of bisphosphonates was chosen for this study. Risedronate sodium exhibited the weakest cytotoxic activity among all tested compounds (The cytostatic drugs chosen for this study are routinely used in canine and human oncology. Doxorubicin showed the strongest cytotoxicity against OS cell cultures (D-17 and U-2 OS), as its ECompounds .p < 0.05) and at its dose of 100 µg/mL the cytotoxic effect of doxorubicin was the strongest (p < 0.05) in both lines. The canine cell line turned out to be more susceptible to the combination of risedronate sodium and doxorubicin than the human one. Cisplatin together with risedronate sodium exerted a significant (p < 0.05) cytotoxicity against the tested cell lines, which was not the case when the substances were used at the same doses but separately. Combined use of 1 µg/mL cisplatin and 100 µg/mL risedronate sodium resulted in the strongest cytotoxic effect and the viability of canine and human cells was 13.44 ± 3.58 and 5.65 ± 4.28%, respectively. We found significant differences in the cytotoxicity between the canine and human OS cell lines after joined use of carboplatin and risedronate sodium. The viability of canine OS cells treated with 1 µg/mL carboplatin and 100 µg/mL risedronate sodium reached 37.2 ± 3.75%. Even though it was lower than after separate administration of the drugs, the difference was not of statistical significance (p > 0.05). In the case of the U-2 OS cell line, the combination of 1 µg/mL cisplatin and 100 µg/mL risedronate sodium in comparison with separate administration of the drugs showed a significant difference and cell viability was 27.75 ± 3.84% (p < 0.05). The tested cell lines were more susceptible to the combination etoposide and risedronate sodium than to their separate administration. The cytotoxic effect depended rather on the concentration of risedronate sodium than that of etoposide. The strongest cytotoxicity was observed for 100 µg/mL of risedronate sodium and 1 µg/mL of etoposide. The viability of the canine OS cell line was 21.84 ± 2.99%, and highly similar to that of the human OS cell line that reached 23.91 ± 4.14%. These values were 50% lower than for the drugs used alone at the same concentrations.The combined administration of selected doses of doxorubicin, cisplatin, carboplatin, etoposide and risedronate sodium in both studied OS cell lines is presented in p < 0.05)  and risedronate sodium may be described as synergy. The strongest apoptosis in both cell cultures was caused by 0.01 µg/mL doxorubicin combined with 100 µg/mL risedronate sodium or 1 µg/mL cisplatin and the same dose of risedronate sodium. The apoptosis rate in the cultures treated with these combinations was 89.21 ± 4.07% and 88.70 ± 6.67% for the canine OS cell line, and 80.91 ± 2.83% and 94.66 ± 4.86% for the human OS cell line, respectively ( < 0.05) .p < 0.05). This was also particularly visible for viability that dropped to 0.00% for canine and 0.01% and 1.03% for human OS cells, following administration of cisplatin at 50 and 20 µg/mL [The study investigated the cytotoxicity of cisplatin, carboplatin, doxorubicin and etoposide against D-17 and U-2 OS cell lines. All the investigated compounds are routinely used in oncological therapy of neoplasms in humans and animals ,31. The 20 µg/mL .50 in both cases, proved similar susceptibility of the evaluated cell cultures to these cytostatic drugs. The two remaining compounds, carboplatin and etoposide, exhibited lower cytotoxicity, as the viability of the tested cell lines reached 34.25 ± 3.92 and 47.04 ± 1.43% and 10.08 ± 5.26 and 24.44 ± 3.83% [Moreover, Poradowski  showed c ± 3.83% , respect50, and the cells of canine OS seemed more susceptible to this drug (EC50 = 6.45 ± 0.2 µg/mL) than human OS cells (EC50 = 27.5 ± 2.3 µg/mL) (p < 0.05). A reverse pattern was observed for the canine and human OS after treatment with etoposide, where human cells showed greater susceptibility (EC50= 2.72 ± 0.51 µg/mL) than canine ones (EC50= 6.27 ± 0.31 µg/mL) (p < 0.05). Following treatment with carboplatin and etoposide, cell viability and EC50 were lower than after the administration of cisplatin and doxorubicin, which proved stronger activity of the latter two drugs against the evaluated cell lines. Several protocols of OS treatment in humans and animals are based on the combined administration of cisplatin and doxorubicin or on a synergy between these and other cytotoxic drugs  [Carboplatin showed higher ECsfamide) ,34.p < 0.05). Risedronate sodium at 100, 10 and 1 µg/mL decreased the viability of canine and human osteosarcoma cell lines to 53.38 ± 1.46 and 49.56 ± 0.7%, 97.08 ± 3.32 and 74.92 ± 4.01%, 100 ± 3.56 and 94.56 ± 3.52%, respectively. The viability of the investigated cell lines and the concentration of risedronate sodium showed a negative correlation that reached p < 0.05, r = 0.42 and p < 0.05, r = 48 for D-17 and U-2 OS, respectively. A comparison of EC50 for canine  and human  OS cell line demonstrated that human OS cells were more susceptible to risedronate sodium than canine OS cell cultures (p < 0.05) [Summarizing, cisplatin alone showed the strongest cytotoxicity against both human and canine OS cell lines, but at higher doses than doxorubicin. The latter was also twice as effective against human than canine OS cell lines. Finally, carboplatin and etoposide brought about the weakest cytotoxic effect, with carboplatin being more effective toward canine and etoposide toward human OS cell lines. The last evaluated compound was risedronate sodium (a bisphosphonate of the third generation), used for treating bone tissue metabolic disorders in humans. The potential cytotoxicity of bisphosphonates suggests their possible use in supporting the activity of cytotoxic drugs in the therapy of neoplasms, including myeloma multiplex or OS ,35,36,37 < 0.05) . Similar < 0.05)  reported < 0.05)  studies 50 in the cell cultures exposed to all tested compounds indicated the strongest cytotoxicity of doxorubicin and cisplatin against canine and human OS (0.056 ± 0.019 and 0.051 ± 0.003 µg/mL and 2.35 ± 0.43 and 2.38 ± 0.43 µg/mL for p < 0.05). Carboplatin and etoposide exhibited weaker cytotoxic effect in canine OS cell cultures. Human OS was the most resistant to carboplatin (EC50= 27.5 ± 2.3 µg/mL) and etoposide and cisplatin cytotoxicity in this line was comparable with EC50= 2.72 ± 0.51 and 2.38 ± 0.43 µg/mL, respectively. Risedronate sodium alone exerted a significant cytotoxic effect only at higher doses and the cell susceptibility was higher in the human OS line.A study by Farese at al.  describeosteosarcoma cell lines (U-2 OS and D-17). In our earlier work [p < 0.05) [The study aimed at investigating a combined activity of risedronate sodium and other cytotoxic drugs in ier work , we demo < 0.05) . Therefoosteosarcoma cell lines. Similarly, as in an earlier study by Poradowski et al. [The combinations of the cytostatic drugs and risedronate sodium exhibited strong anticancer activity in the tested i et al. , potentii et al.  in U-2 Oi et al.  in an esosteosarcoma (LM-8) cell culture and proved its concentration dependence. Apart from risedronate sodium, other bisphosphonates, such as zoledronate sodium and alendronate sodium, strongly promote apoptosis in both tested cell lines [p < 0.05. Still, the activity of these combinations was lower than that presented by doxorubicin and cisplatin combined with risedronate sodium. The pro-apoptotic activity of all compound combinations confirmed the results achieved in the MTT assay.Our study revealed a very strong apoptotic effect of risedronate sodium in D-17 and U-2 OS cell lines. Murayama et al.  also obsll lines . A consiThe study results seem to support the hypothesis that both pro-apoptotic and cytotoxic activity of the investigated cytostatic drugs can be increased by risedronate sodium. A similar synergy between zoledronate sodium and doxorubicin was reported by Neville-Webbe et al.  in the eFinally, all results achieved in vitro must be verified during in vivo experiments. The concentrations of the investigated compound were chosen on the basis of accessible literature. Even though they may be achieved in the blood plasma of living animals or humans, only the studies in experimental animals can confirm or disprove the synergistic effects of risedronate sodium combined with cytostatic drugs.The supporting role of risedronate sodium in the cytotoxic effects provided by drugs used in the treatment of OS and other neoplasms was demonstrated in vitro in D-17 and U-2 OS cell lines. Our analysis can confirmed the hypothesis of a synergistic character of these combinations of cytostatic compounds. Further studies on normal human and canine cell lines and subsequent in vivo experiments are needed to verify the presented in vitro outcomes.


Cancer cells have an increased need for glucose and, despite aerobic conditions, obtain their energy through aerobic oxidation and lactate fermentation, instead of aerobic oxidation alone. Glutamine is an essential amino acid in the human body. Glutaminolysis and glycolysis are crucial for cancer cell survival. In the therapy of estrogen receptor α (ERα)-positive breast cancer (BC), the focus lies on hormone sensitivity targeting therapy with selective estrogen receptor modulators (SERMs) such as 4-hydroxytamoxifen (4-OHT), although this therapy is partially limited by the development of resistance. Therefore, further targets for therapy improvement of ERα-positive BC with secondary 4-OHT resistance are needed. Hence, increased glucose requirement and upregulated glutaminolysis in BC cells could be used. We have established sublines of ERα-positive MCF7 and T47D BC cells, which were developed to be resistant to 4-OHT. Further, glycolysis inhibitor 2-Deoxy-D-Glucose (2-DG) and glutaminase inhibitor CB-839 were analyzed. Co-treatments using 4-OHT and CB-839, 2-DG and CB-839, or 4-OHT, 2-DG and CB-839, respectively, showed significantly stronger inhibitory effects on viability compared to single treatments. It could be shown that tamoxifen-resistant BC cell lines, compared to the non-resistant cell lines, exhibited a stronger reducing effect on cell viability under co-treatments. In addition, the tamoxifen-resistant BC cell lines showed increased expression of proto-oncogene c-Myc compared to the parental cell lines. This could be reduced depending on the treatment. Suppression of c-Myc expression using specific siRNA completely abolished resistance to 4OH-tamoxifen. In summary, our data suggest that combined treatments affecting the metabolism of BC are suitable depending on the cellularity and resistance status. In addition, the anti-metabolic treatments affected the expression of the proto-oncogene c-Myc, a key player in the regulation of cancer cell metabolism.  In human tissue, two estrogen receptors (ERs) can basically be distinguished from one another, ERα and ERβ. Both have the same basic structure but are expressed in different tissues and show different effects. While ERα is mainly found in the breast, ERβ is expressed in bone, endometrium and blood vessels . Tamoxif2, pyruvate, lactate, alanine and citrate [4+). In the next step, glutamate dehydrogenase turns glutamate into α-ketoglutarate, which then can feed the citric acid cycle. Alternatively, glutamate can be converted by alanine aminotransferase or by aspartate aminotransferase into alanine and aspartate, which are essential for nucleic acid synthesis. Malate produced in the intermediate step of the citric acid cycle can be converted into pyruvate or lactate. As part of glutaminolysis, this opens up another process of energy generation in addition to glycolysis [Selectivity is a cornerstone in cancer therapy. In order to achieve effective damage of tumor tissue without side effects, it is important to recognize biological differences between normal cells and tumor cells. Normally, aerobic glycolysis takes place under oxygen conditions and anaerobic glycolysis under oxygen deficiency. In aerobic glycolysis, glucose is metabolized into carbon dioxide, water and ATP via citrate cycle and mitochondrial respiratory chain. Anaerobic glycolysis works through the fermentation of pyruvate into lactate with a significantly lower gain of ATP . Cancer  citrate . After bycolysis . It has ycolysis ,16. The ycolysis .This knowledge represents a potential therapeutic target in the way that glutaminolysis is inhibited. The glutaminase inhibitor CB-839 interferes with the first step of glutaminolysis by selectively and irreversibly binding the enzyme glutaminase . Increas2 in air as previously described [The human breast cancer cell lines MCF7 and T47D were obtained from the American Type Culture Collection . To guarantee the identity of the cell lines over the years, the cells were expanded after purchase and aliquots were stored in liquid nitrogen. Every half-year, new frozen stock was opened and expanded to carry out the experiments. Tamoxifen-resistant sublines MCF7-TR and T47D-TR were developed as described in detail , and theescribed .4-hydroxytamoxifen (4-OHT) and 2-Deoxy-D-glucose (2-DG) were purchased from Sigma. Glutaminase inhibitor CB-839 was purchased from Selleckchem .2. Final concentrations of 4-OHT, 2-DG, CB-839 and the combinations of the agents are given in the results section. Cell number was determined by a colorimetric assay using Alamar Blue . The optical density (OD) of the reduced dye was assessed at 570 versus 630 nm after 4 h at 37 °C.A total of 12,500 cells per well were plated into 96-well plates  in 100 μL Dulbecco’s Modified Eagle’s Medium/5% fetal calf serum  without phenol red, 2 mM glutamine, 50 U/mL penicillin/streptomycin, 2.5 μg/mL amphotericin B and 1% non-essential amino acids. After cell attachment, 100 μL medium, 100 μL 4-OHT/medium solution, 100 μL 2-DG/medium solution, 100 μL CB-839/medium solution or 100 μL solution with combination treatments were added to the wells and incubated for 96 h at 37 °C, 5% COCells were treated for 48 h with or without 4-OHT, 2-DG, CB-839 or the combinations of the agents. Then they were washed once with PBS and mitochondrial membrane potential was measured using the JC-1 mitochondrial membrane potential detection kit according to the instructions of the manufacturer .In Western Blot analysis, cells were lysed in cell lytic M buffer (Sigma) supplemented with 0.1% phosphatase-inhibitor (Sigma) and 0.1% protease-inhibitor (Sigma). Isolated proteins (40 µg) were fractioned using 12% SDS gel and electro-transferred to a polyvinylidene difluoride membrane . Primary antibodies against c-Myc 1:10,000  and GAPDH 1:2000  were used. The membrane was washed and incubated in horseradish peroxidase-conjugated secondary antibody . Antibody-bond protein bands were assayed using a chemiluminescent luminol enhancer solution . For visualization and quantification, the bands were scanned using a C-DiGit Blot Scanner .5 cells/mL) and T47D-EMT (2.5 × 105 cells/mL) were seeded in 2 mL of MEM with 10% FBS (-P/S) in 25 cm2 cell culture flask. The cells were transiently transfected with siRNA specific to c-Myc  in OPTI-MEM I medium  with a siRNA transfection reagent . A non-targeting siRNA was used as control . After an incubation period of 6 h, MEM supplemented with 20% FBS and 20% penicillin/streptomycin was added.Breast cancer cell lines MCF7-EMT .We analyzed, whether the therapy of ERα-positive human breast cancer cell lines MCF7 and T47D with 4-OHT can be optimized by an anti-metabolism therapy using 2-DG and/or CB-839 and whether differences to the corresponding tamoxifen-resistant sublines MCF7-TR and T47D-TR could be observed.First, the effects of 4-OHT, 2-DG and CB-839 on cell viability were determined separately on the cell lines MCF7, MCF7-TR, T47D and T47D-TR using three different doses of each drug.The MCF7 cell line  showed aUnder therapy with 4-OHT, the T47D cell line  showed aThere was also a significant inhibition of viability with the T47D-TR subline detectable compared to the untreated control . After tThe treatment of the T47D cell line  with 5 μTreatment with the combination of 4-OHT and 2-DG resulted in a significant reduction in the viability of MCF7 cells  comparedThe MCF7-TR cell line  showed aDuring the treatment with the combination of 2-DG and CB-839, a significantly stronger viability-inhibiting effect was observed compared to the treatments with 4-OHT, CB-839, 4-OHT/2-DG and 4-OHT/CB-839. Similarly, the combination of all three drugs showed a significantly stronger inhibition of viability compared to the treatments with 4-OHT, CB-839, 4-OHT/2-DG and 4-OHT/CB-839.The single treatments of T47D cells  with 2-DTreatment of T47D cells  with theTreatment of T47D-TR cells  with 2-DThe combination therapy of MCF7 cells  with 4-OTreatment of the MCF7-TR cell line  with theAfter the treatment of T47D cells  with 4-OThe T47D-TR cell line  showed aThe combination treatment 4-OHT/2-DG of T47D-TR cells  led to ap < 0.05 vs. T47D).In the next step, we analyzed the role of the proto-oncogene c-Myc during the treatments with 4-OHT, 2-DG, CB-839 and their combinations. First, it was checked to what extent c-Myc was expressed by the four untreated cell lines MCF7, MCF7-TR, T47D and T47D-TR in comparison to one another A,B. The To confirm the impact of c-Myc suppression on MCF7, MCF7-TR, T47D and T47D-TR breast cancer cells, we assessed whether knock down of c-Myc expression using specific siRNA leads to reduced viability . SuppresIn the next step, we examined the effects of the treatments on the expression of c-Myc. Significant downregulation of c-Myc in the MCF7 cell line  was demoThe main question of this work was whether the therapy with low-dose 4-OHT can be optimized by an anti-metabolistic therapy using 2-DG and/or CB-839 and whether differences compared to the tamoxifen-resistant sublines could be observed. In addition, the effects of these treatments on the expression of c-Myc should be investigated.It could be shown, that therapy with 4-OHT resulted in a dose-dependent reduction of viability of both parental, ERα-positive breast cancer cell lines. Treatment of the tamoxifen-resistant sublines showed no or a less pronounced effect on viability. The fact, that treatment of the two sublines with the highest dose of 4-OHT resulted in a significant inhibitory effect on viability suggests that secondary anti-estrogen receptor resistance was not fully developed at this point. 2-DG also led to a dose-dependent reduction in viability, both in the tamoxifen-sensitive and in the tamoxifen-resistant cell lines. In comparison, it could be observed that the tamoxifen-resistant cell lines in both cases showed greater inhibition of viability compared to their parental cell lines at a lower concentration (2.5 mM 2-DG). At a higher concentration (5 mM 2-DG), interestingly, the effect on the parental cell lines was stronger compared to their tamoxifen-resistant cell lines.A dose-dependent profile of action could be shown under therapy with CB-839 in all four cell lines. However, compared to its parental cell line MCF7, the tamoxifen-resistant cell line MCF7-TR showed a stronger inhibition of viability under the highest CB-839 concentration (10 µM CB-839). The two cell lines T47D and T47D-TR behaved similarly with regard to the action profile. The strongest effects could be achieved with the MCF7 cell line treated with the highest concentrations of 4-OHT or 2-DG. In comparison, the associated tamoxifen-resistant cell line showed the strongest effect under the highest concentrations of the metabolism inhibitors 2-DG and CB-839. The T47D cell line presented the strongest inhibitory effects on cell viability under treatment with the highest concentrations of 4-OHT and 2-DG, whereas the associated tamoxifen-resistant cell line showed the strongest inhibitory effect among the highest concentrations of the metabolism inhibitors 2-DG and CB-839. This initially resulted in the observation that the parental cell lines MCF7 and T47D had the greatest effects under the 4-OHT and 2-DG therapy compared to the other treatments. In contrast, the two tamoxifen-resistant cell lines MCF7-TR and T47D-TR presented strong inhibitions of viability under treatment with the metabolism inhibitors 2-DG and CB-839. In summary, 2-DG could be evaluated as an effective treatment for all four cell lines. 4-OHT showed little or no efficacy in the tamoxifen-resistant cell lines. Here, CB-839 proved to be effective.Gross et al. showed that CB-839 has a proliferation-inhibitory effect on breast cancer cells from different subtypes, but not on the T47D cell line at low concentrations of 1 µM . This coOur data confirm this presumption to the extent that the parental, non-resistant cell lines MCF7 and T47D only showed a significant reduction in viability above 5 mM 2-DG, the tamoxifen-resistant cell lines MCF7-TR and T47D-TR already below concentrations of 2.5 mM 2-DG. However, in both cell lines, the tamoxifen-resistant cell lines showed a weaker inhibition at the highest concentration (5 mM 2-DG) compared to their parental lines. Subsequently, the effects of the various treatments in combination were examined in more detail and compared in a viability assay. In the case of the MCF7 cell line, the combination treatment of the two metabolism inhibitors, as well as the triple combination, showed the strongest viability inhibition compared to the other treatment alternatives. The associated tamoxifen-resistant cell line showed, in addition to an already strong inhibition of viability under the 2-DG therapy alone, a comparatively strong inhibition by the combined metabolism inhibitors and by the triple combination. In principle, the viability inhibition of the tamoxifen-resistant cell line MCF7-TR was more pronounced than of the parental cell line MCF7. The T47D cell line reacted under the combined metabolism inhibition as well as the triple combination most strongly in a viability-inhibiting manner in contrast to the other treatments. In comparison, the T47D-TR cell line showed the strongest viability-inhibiting effects among the triple combination. Overall, it can be summarized that the combination therapies presented better effectiveness in all cell lines compared to their individual therapy counterparts. In the viability assay, the tamoxifen-resistant cell lines showed stronger viability-inhibiting effects on all combination treatments compared to the parental cell lines. It was also observed that the combined inhibition of metabolism was superior to the combined tamoxifen therapy in all cell lines. The combination of the two metabolism inhibitors 2-DG and CB-839 showed a significantly stronger inhibition of cell viability in all four cell lines compared to the individual treatments. Through the triple combination therapy, a synergistic-supportive inhibitory effect could be produced. In the viability assay alone, therapy with low-dose 4-OHT showed a significantly better viability-inhibiting effect in the tamoxifen-resistant cell lines when combined with CB-839 compared to the respective individual treatments. The combination of 4-OHT and 2-DG failed with no significantly stronger inhibition of viability in any of the four cell lines.In order to consider potential effects on healthy breast tissue, studies were also carried out with the normal breast epithelial cell line MCF-10A (not shown). Whereas these cells exhibit a non-significant inhibitory effect of 004-OHT on cell growth, the anti-metabolic agents 2-DG and CB-839 alone or in combination did not show any effects. Furthermore, there was no change in the effects of 4-OHT if the cells were treated in combination with 2-DG and/or CG-839. Tamoxifen has been reported to have beneficial effects on normal breast tissue. Tamoxifen affects the normal breast epithelium and its adhesiveness, which probably contributes to the clinically observed decrease of breast density and thus could lead to a potentially reduced risk of developing breast tumors . The resTo better classify the inhibition of viability with regard to a more precise mechanism among the various treatments, an apoptosis assay was carried out. It could be shown that to a large extent, there was a correlation between the viability assay and apoptosis. In the MCF7 cell line, inhibition of the mitochondrial membrane potential was observed under the triple combination, but also under the combinations of 4-OHT and 2-DG as well as the metabolism inhibitors. The same was found in the associated tamoxifen-resistant MCF7-TR cell line, as well as the T47D and T47D-TR cell lines. It was observed that in three of the four cell lines  a significantly strong inhibition of the mitochondrial membrane potential was caused by the combination treatment 4-OHT + 2-DG at the time of measurement was performed. This effect of apoptosis induction was not reflected in the viability behavior and is most likely to be explained by the sensitivity of the method itself.To investigate a possible connection between the effects of the various anti-metabolic treatments on cell viability and the role of the oncogenic protein c-Myc in the regulation and control of cancer cell metabolism , expressHowever, if various processes such as a p53 mutation or the anti-apoptotic protein Bcl-2 lead to an inhibition of apoptosis, the system is out of balance and tumor development is guaranteed . It is pWatson et al. have already demonstrated c-Myc amplification in MCF7 and T47D cell lines . In our We could also show that c-Myc downregulation was partially significant under almost all treatments, especially under the combination treatments. The fact that suppression of c-Myc expression using specific siRNA leads to the loss of tamoxifen-resistance in tamoxifen-resistant cell lines MCF7-TR and T47D-TR supports the important role of c-Myc in this context. This may confirm the connection between the active ingredients and c-Myc. Treatment with 4-OHT alone was an exception in the case of the tamoxifen-resistant cell lines; there was no down-regulation of the c-Myc. This shows that the secondary tamoxifen resistance must have an impact on c-Myc expression.Combination treatments of 4-OHT with CB-839, with 2-DG and CB-839 as well as the triple combination of 4-OHT, 2-DG and CB-839 have significantly stronger inhibitory effects in vitro compared to the individual treatments with 4-OHT, 2-DG or CB-839 alone. However, none of the cell lines showed a therapy optimization compared to the individual treatments with the combination of 4-OHT and 2-DG. The viability-inhibiting effects were largely reflected in the induction of apoptosis. In addition, a downregulation of the proto-oncogene c-Myc could be observed during the treatments. Furthermore, after the suppression of c-Myc expression using specific siRNA the loss of tamoxifen-resistance of the tamoxifen-resistant BC cells was observed. These results support the major role of c-Myc in the regulation of tamoxifen resistance and cancer metabolism. The cell lines MCF7 and T47D, due to their properties as ER- and PR-positive epithelial, non-invasive cell lines, represent an in vitro model of breast cancer of the subtype luminal A and thus the most common form of BC. In practice, tamoxifen is an established therapy recommendation for this subtype. However, therapy becomes more difficult due to the development of side effects and occurrences of secondary resistance. In this work, it was shown that the therapy with tamoxifen alone in the cell lines MCF7 and T47D could be optimized by combination with the glutaminase inhibitor CB-839. In vivo, it would make sense to evaluate this combination, especially with the purpose of reducing the concentrations of the individual substances in order to counteract the possible development of side effects. The development of secondary tamoxifen resistance is a common problem in practice. With the tamoxifen-resistant cell lines MCF7-TR and T47D-TR in connection with their parental lines, an in vitro model was available to compare the different treatments. It could be shown that the tamoxifen-resistant cell lines in particular reacted more sensitively to the combined metabolic inhibition in comparison to their parental cell lines. An in vivo evaluation of this combination would be valuable. With the T47D-TR cell line, a significantly stronger effect compared to all other treatments was observed even using the triple combination treatment. Therefore, it would be interesting to evaluate whether tamoxifen resistance could be overcome by treatment with the combination of both inhibitors.


Aim: Innate resistance to the CHK1 inhibitor prexasertib has been described, but resistance mechanisms are not understood. We aimed to determine the role epidermal growth factor receptor (EGFR) plays in innate resistance to prexasertib in triple negative breast cancer (TNBC).Methods: Using a panel of pre-clinical TNBC cell lines, we measured the sensitivity to prexasertib. We examined the effect activation of EGFR had on prexasertib sensitivity. We measured the synergy of dual blockade of EGFR with erlotinib and CHK1 with prexasertib in TNBC cell lines and xenografts.Results: EGFR overexpression and activation increased resistance to CHK1 inhibition by prexasertib. EGFR promoted the phosphorylation of BCL2-associated agonist of cell death (BAD), inactivating its pro-apoptotic functions. Inhibition of EGFR reversed BAD phosphorylation, increasing sensitivity to prexasertib.Conclusion: The use of prexasertib as a monotherapy in TNBC has been limited due to modest clinical responses. We demonstrated that EGFR activation contributes to innate resistance to prexasertib in TNBC and potentially other cancers. EGFR expression status should be considered in clinical trials examining prexasertib’s use as a monotherapy or combination therapy. The lack of these targetable molecules for hormone and growth factor therapy limit current treatment options for TNBC.Triple negative breast cancer (TNBC) is an aggressive form of breast cancer associated with poor prognosis and metastasis. TNBC is characterized by the lack of estrogen receptor  proteins, such as ATM, ATR, and CHK1, as molecular targets in aggressive cancers such as TNBC-5. CHK1 plays a critical role in response to DNA damage and is an essential effector in the regulation of replication. Inhibitors of CHK1 alter DNA damage response and abrogate S and G2-M cell cycle checkpoints to promote replication stress, induce double strand breaks, and promote cell death through mitotic catastrophe in highly proliferating cells. In the last several years, CHK1 inhibitors, such as prexasertib (LY2606368), have shown success in promoting cell death as a monotherapy in high grade serous ovarian cancer, squamous cell carcinoma, and neuroblastoma,7-9.Interestingly, activation of ATR/CHK1 has been observed as a method of stabilizing the replication fork and promoting resistance to PARP inhibitors. Several reports have demonstrated that prexasertib is effective, particularly as combination therapies in TNBC-13. However, as a monotherapy, prexasertib has only shown modest activity in the treatment of TNBC (NCT02203513). Modest clinical activity may be due to innate or acquired resistance to prexasertib, which is poorly understood.The lack of molecular targets in TNBC has prompted investigations into CHK1 inhibitors as single-agent or combination therapieset al. described innate and acquired resistance to prexasertib in sarcoma xenografts. Prexasertib-resistant tumors showed higher levels of the anti-apoptotic protein BCL-xL and increased phosphorylation along the PI3K and MAPK signaling pathways. Specifically, highly activated AKT, MEK1/2, and ERK1/2 were observed in resistant tumors. Increased RAS/MEK/ERK activity has also been reported in response to CHK1 inhibition in other cell lines,16. However, combinations of MAPK or PI3K inhibitors with prexasertib were insufficient to overcome acquired resistance in sarcoma xenografts.In 2019, Lowery -19. Therefore, we examined EGFR expression and inhibition in TNBC preclinical cell lines to determine if EGFR promoted resistance to prexasertib and if inhibition of EGFR could enhance the anti-tumor activity of prexasertib in TNBC tumors.Innate or acquired resistance to prexasertib may arise from mechanisms that drive cell proliferation and stabilize the replication fork. An upstream regulator of both MAPK and PI3K pathways is the epidermal growth factor receptor (EGFR). EGFR overexpression or activation stimulates RAS/MAPK and, to a lesser extent, PI3K/AKT/mTOR signaling, driving cell proliferation and potentially bypassing prexasertib-induced replication stress. EGFR overexpression is observed in ~50% of TNBC tumors and is associated with poor overall survivalThe TNBC cell lines HCC1806, HCC1937, MDA-MB-157 (MDA-157), MDA-MB-231 (MDA-231), and MDA-MB-468 (MDA-468) were purchased from the American Type Culture Collection . MX-1 was purchased from the NCI repository. All cell lines were purchased within the previous 24 months and passaged < 15 times for all experiments Cytotoxicity of monolayer and 3D cultures were determined with a cell viability assay, CellTiter-Glo and CellTiter-Glo 3D . For monolayer experiments, HCC1806, HCC1937, MDA-157, MDA-468, and MX-1 were plated at 5000 cells per well in clear-bottomed white 96-well plates. MDA-231 cells were plated at 2500 cells per well in clear-bottomed white 96-well plates. The cells were allowed to attach for two days, and then they were treated with increasing concentrations of prexasertib , erlotinib , or both agents. The cells were exposed continuously for four days, and the viability was assessed with CellTiter-Glo. For EGF stimulation, MDA-231 or MDA-468 were treated with EGF  at 50 or 500 nmol/L at the same time as the indicated concentrations of prexasertib were added. Cells were exposed continuously for four days and viability assessed with CellTiter-Glo.For spheroid growth, clear-bottomed white 96-well plates were coated with 50 µL of Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix . MDA-231 and MDA-468 cells were then seeded at 5000 cells per well on top of the gel layer in DMEM with 2.5% Matrigel. The cells were grown for four days to allow spheroids to form. The cells were then treated with increasing concentrations of prexasertib, erlotinib, or both agents. Cells were exposed continuously for four days, and the viability was assessed with CellTiter-Glo 3D.50) using software .The assay reagent (CellTiter-Glo or CellTiter-Glo 3D) was added to plates and incubated according to the manufacturer’s instructions. Luminescence was read on a multimodal plate reader . All viability assays were performed with technical triplicates over three biological replicates. The results were normalized to values for cells exposed to vehicle control and graphed to generate half-maximal inhibitory concentration  values were analyzed using the readily available CompuSyn software. Cells were grown in 10-cm dishes and cultured to 70%-80% confluence. Cells were rinsed with phosphate-buffered saline, scraped, stored overnight at -80 °C, and lysed. Lysates were separated on a 4%-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel  and transferred to a nitrocellulose membrane. The membrane was probed with antibodies diluted in 5% nonfat dry milk in Tris-buffered saline  and 0.1% Tween 20 (Fisher Scientific) and raised against reagents for immunoblot Immunoblotting was performed as described previouslyAll procedures were performed in accordance with guidelines approved by the University of South Alabama’s Institutional Animal Care and Use Committee (IACUC). All animals were allowed access to food and water ad libitum and received veterinary care.6 MDA-231 cells or 107 MDA-468 cells 1:1 in low growth factor Matrigel. Nineteen days after MDA-231 tumor implantation and twenty-six days after MDA-468 tumor implantation, mice were dosed with the vehicle, prexasertib (dissolved in 40% captisol and sterile water) alone, erlotinib (dissolved in 4% captisol and sterile water) alone, or a combination of prexasertib and erlotinib. Prexasertib was administered via subcutaneous injection [10 mg/kg body weight (BW)] twice daily for three days, followed by a four-day rest. Erlotinib was administered via oral gavage (50 mg/kg BW) daily. Dosing amounts and schedules were selected based on experiments by Lowery et al. looking at combination exposures with prexasertib and other small molecule inhibitors and reports examining erlotinib response of TNBC xenografts,9,22,23. Animals were treated for four weeks. Tumor volumes were calculated using the formula V = (L × W × W)/2, where V is volume, L is length, and W is width. Body weights were analyzed twice weekly, and animals were observed for signs of distress or pain and sacrificed when appropriate.Forty athymic nude mice (Charles River) were implanted subcutaneously with 1050 values ± standard error of the mean (SEM) were determined from at least three biological replicates. Tumor volumes, tumor weights, and body weights are presented as mean ± SEM. All values were evaluated with one-way analysis of variance (ANOVA), and means were compared with Dunnett’s post hoc test or Tukey’s post hoc test. Statistical significance was defined by *P < 0.05, **P < 0.01, and ***P < 0.001.Mean IC50, 5.7 nmol/L), while MDA-468 cells were highly resistant .We first examined the sensitivity of a panel of TNBC cell lines to prexasertib . MDA-468 also contained the highest expression level of EGFR. We examined AKT activation in the TNBC cell line panel and observed high activation of AKT in the MDA-468 and MX-1 cell lines. The activation of other DDR proteins in these cell lines is shown in Supplementary Figure 1.Immunoblot analysis of the BRAF/MEK/ERK signaling pathway in the TNBC cell lines showed mixed activation of this pathway in the prexasertib-sensitive cell lines 50 shifting from 17 to 61 nmol/L Given the overexpression of EGFR in the MDA-468 cell line and the BRAF/MEK/ERK pathway stimulation, we examined whether stimulation by EGF would reduce the sensitivity of MDA-231 to prexasertib P < 0.001 compared to control), 52 ± 5% (P < 0.001 compared to control), and 47% ± 5% (P < 0.001 compared to control) for 1, 2, and 10 µmol/L, respectively With EGF stimulating prexasertib resistance in the MDA-231 cells, we tested the synergy of EGFR inhibition with prexasertib. Given that efficacy can differ between monolayer culture and three-dimensional (3D) culture, we tested the combination of erlotinib and prexasertib in spheroids of MDA-231 and MDA-468 . A reduction in activated AKT was also observed in the prexasertib-resistant MDA-468.Immunoblotting of MDA-231 and MDA-468 treated with prexasertib, erlotinib, and both prexasertib and erlotinib showed that the co-exposures reduced the phosphorylation of BCL2-associated agonist of cell death (BAD), releasing the pro-apoptotic protein from 14-3-3 sequestration [Figure 5]Immunoblotting of EGF stimulated MDA-231 cells also confirmed that EGF activation increased phosphorylation of BAD at serine 112 (S112), reducing apoptotic activity and promoting resistance to prexasertib in vivo efficacy of co-dosing erlotinib and prexasertib in MDA-231 and MDA-468. MDA-231 is a commonly used preclinical model cell for TNBC with representative sensitivity to prexasertib 3 to 145 ± 77.3 mm3, P = 0.026, 3) and erlotinib alone (223 ± 101 mm3). No significant decrease in body weight was observed during monotherapy or combination therapy in either xenograft model [Supplementary Figure 2].Finally, we examined the Immunoblot of the final MD-468 tumors showed a trend for reduced EGFR activity in tumors treated with both prexasertib and erlotinib ,7,9. CHK1 inhibitors’ efficacy as monotherapy in TNBC has been limited by modest clinical responses. Recent studies have described innate and acquired resistance to prexasertib that could help develop biomarkers to stratify patients for prexasertib monotherapy treatment.Molecular targeting has significantly enhanced survival for hormone and growth factor positive breast cancers. TNBC has remained challenging to treat because effective molecular targets have not been identified. CHK1 inhibitors such as prexasertib have shown efficacy as a monotherapy in difficult to treat tumor types such as neuroblastoma and high-grade serous ovarian tumorset al. noted that the colorectal cancer cell line HCT116 and pancreatic carcinoma PANC-1 showed no sensitivity to prexasertib. In 2019, Lowery et al. demonstrated the acquired resistance to prexasertib in a rhabdomyosarcoma xenograft and innate resistance in Ewing’s sarcoma and osteosarcoma cell lines. Acquired resistance was characterized by the activation of the PI3K and MAPK pathways, but there was no characterization of the pathways involved in innate resistance. More recently, Nair et al. observed acquired resistance in BRCA wild-type high-grade serous ovarian cancers and attributed resistance to downregulation of CDK1/cyclin B1 prolonging G2 and delaying mitotic catastrophe. Contributions of the PI3K and MAPK pathways were not examined.When characterizing prexasertib in 2015, King BRCA wild-type and BRCA mutated TNBC cell lines, we noted innate resistance to prexasertib in the MDA-468 cell line et al. for acquired resistance et al. observed no reversal of prexasertib resistance with MAPK inhibitors, we examined EGFR’s role in innate prexasertib resistance.While acquired resistance may contribute to the modest clinical response observed with prexasertib in TNBC, innate resistance in TNBC tumors may also be a driving factor. We currently lack biomarkers for intrinsic resistance to prexasertib in TNBC and other cancers. Examining a panel of Stimulation of EGFR by EGF in the MDA-231 cells resulted in increased resistance to prexasertib in vivo, although there was no apparent benefit to combination treatment in MDA-231 xenografts in vivo experiments is likely due to higher dosing of prexasertib and erlotinib in the in vivo experiments. Erlotinib dosing for the xenograft models was selected based on existing literature and known sensitivity of TNBC cell lines,23. Prexasertib dosing was also selected based on existing literature,9. The synergy of the 3D experiments and the high tumor reduction observed in our animal experiments suggest future dosing experiments should be conducted to optimize the prexasertib to erlotinib ratios to minimize side effects, while maintaining tumor reduction. Myelosuppression is commonly a dose-limiting factor for CHK1 inhibitors in the clinical setting. We did not see significant toxicity or weight loss with prexasertib, erlotinib, or combination treatment in either xenograft model [Supplementary Figure 2]. However, further dose optimization of the combination treatment could reduce adverse effects while maintaining tumor reduction. Our results are also consistent with those of Zeng et al., who reported enhanced cell killing and tumor targeting with cetuximab and prexasertib in head and neck squamous cell carcinoma. Prexasertib resistance was not reported in their xenograft models.Tumor xenografts of MDA-468 confirm the synergistic activity of prexasertib and erlotinib -19,27. Our data indicate that activation or overexpression of EGFR contributes to innate resistance to prexasertib in TNBC and may contribute to the modest clinical efficacy observed in phase I and II trials. Stratification of prexasertib clinical data based on EGFR expression status could offer new insight into the clinical use of prexasertib as a monotherapy for TNBC and other cancers. It should be noted that EGFR overexpression is also observed in HCT116 and PANC-1 cell lines, which have an innate resistance to prexasertib,28,29. Ewing’s sarcoma and osteosarcoma also have a significant percentage of EGFR overexpression, which may contribute to their observed innate resistance to prexasertib,16,30,31. In 2017, Lee et al. noted the complex molecular interactions of the RAS/RAF/MEK/ERK pathways with CHK1 inhibitor sensitivity. The data presented here support EGFR as a biomarker for the use of prexasertib and potentially other CHK1 inhibitors.EGFR overexpression has been noted in at least 50% of TNBCs, which is higher than other breast cancer subtypes


TM to explore the NCI-60 drug databases for compounds with anticancer activities correlating most closely with the mRNA expression of NQO1, a marker for NRF2 pathway activity. Among the >100,000 compounds analyzed, NSC84167, termed herein as NRF2 synthetic lethality compound-01 (NSLC01), was one of the top hits  and selected for functional characterization. NSLC01 selectively inhibited the viabilities of four out of seven conventional pancreatic cancer cell lines and induced dramatic apoptosis in the cells with high NRF2 activation. The selective anticancer activity of NSLC01 was further validated with a panel of nine low-passage pancreatic patient-derived cell lines, and a significant reverse correlation between log(IC50) of NSLC01 and NQO1 expression was confirmed . Notably, screening of a panel of nine patient-derived xenografts (PDXs) revealed six PDXs with high NQO1/NRF2 activation, and NSLC01 dramatically inhibited the viabilities and induced apoptosis in ex vivo cultures of PDX tumors. Consistent with the ex vivo results, NSLC01 inhibited the tumor growth of two NRF2-activated PDX models in vivo  but had no effects on the NRF2-low counterpart. To characterize the mechanism of action, we employed a metabolomic isotope tracer assay that demonstrated that NSLC01-mediated inhibition of de novo synthesis of multiple amino acids, including asparagine and methionine. Importantly, we further found that NSLC01 suppresses the eEF2K/eEF2 translation elongation cascade and protein translation of asparagine synthetase. In summary, this study identified a novel compound that selectively targets protein translation and induces synthetic lethal effects in NRF2-activated pancreatic cancers.Nuclear factor erythroid 2-related factor 2 (NRF2) is aberrantly activated in about 93% of pancreatic cancers. Activated NRF2 regulates multiple downstream molecules involved in cancer cell metabolic reprogramming, translational control, and treatment resistance; however, targeting NRF2 for pancreatic cancer therapy remains largely unexplored. In this study, we used the online computational tool CellMiner Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2), or nuclear factor erythroid 2-related factor 2 (NRF2), is a transcription factor that maintains cellular redox homeostasis by regulating the expression of antioxidant genes, including glutathione peroxidase, thioredoxin 1, thioredoxin reductase, peroxiredoxin 1, and sulfiredoxin 1 . Under oKEAP1, CUL3, or NRF2 can cause constitutive activation of NRF2 perimidine-4-carboxylic acid) was purchased from Sigma. Conventional pancreatic and non-small cell lung cancer cell lines were from American Type Culture Collection, and primary pancreatic cancer cells were established from PDXs in our laboratory as described previously perimidine-4-carboxylic acid) as one of the top hits; its pattern of growth inhibition across the 60 cell lines correlated most closely with NQO1 expression  , whereas HPAF-II, HS776T, and BXPC3 were resistant   were very sensitive to NSLC01, whereas, two KEAP1 wild-type counterparts (HCC193 and H1299) and hTERT-HPNE were resistant to NSCLC-01  or sh-NRF2–88, which has no knockdown effect, did not affect the activity of NSC84167 Fig. .Taken together, these results indicate that the anticancer activity of NSLC01 is correlated with activation of NRF2.To further investigate the mechanism of action of NSLC01, we performed an apoptosis assay using flow cytometry. Exposure of cells to 3 or 10 µM NSLC01 for 48 h induced dramatic apoptosis in sensitive lines but no evident effect in the resistant line Fig. . Western50) of NSLC01  patient-derived pancreatic cancer cell lines established from our PDAC PDXs Fig. .. In MTTThe selective anticancer activity of NSLC01 was further tested using a previously established LTSA method , an ex v 3B,p < 0.01) and PATX124 (p < 0.01). These results were consistent with the ex vivo results  and one resistant PDX (PATX102) to further validate the anticancer activity of NSLC01 in vivo. Compared with vehicle, NSLC01 significantly inhibited tumor growth of two sensitive PDXs, PATX110  which checks protein synthesis by fluorescence imaging without using isotopes. NSLC01 dramatically inhibited protein translation in sensitive ASPC1 cells but not in resistant BXPC3 cells Fig. . In addiOn the basis of these data, we propose the following as the mechanism of action model for NSLC01 Fig. : NSLC01 KRAS mutation upregulates NRF2 expression through activation of MAPK pathway, and indeed, we observed that BXPC3 cell line harboring a wild-type KRAS gene is resistant to NSLC01  of pancreatic cancers, further optimization and formulation of this lead compound may provide a novel biomarker-guided therapeutic approach to treating pancreatic cancer.Supplementary Figure legends.Supplementary Fig. 1.Supplementary Fig. 2.Supplementary Fig. 3.Supplementary Tables.


This study demonstrates differences in cell survival between nanoparticles and cell line. U87 shows the greatest enhancement with gadolinium nanoparticles (2.02 ± 0.36), whereas MCF‐7 cells have higher enhancement with gold nanoparticles (1.74 ± 0.08). Mass spectrometry, however, shows highest elemental uptake with iron oxide and U87 cells with 4.95 ± 0.82 pg of iron oxide per cell. A complex relationship between cellular elemental uptake is demonstrated, highlighting an inverse correlation with the enhancement, but a positive relation with DNA damage when comparing the same nanoparticle between the two cell lines.Materials with a high atomic number (Z) are shown to cause an increase in the level of cell kill by ionizing radiation when introduced into tumor cells. This study uses in vitro experiments to investigate the differences in radiosensitization between two cell lines (MCF‐7 and U87) and three commercially available nanoparticles  irradiated by 6 MV X‐rays. To assess cell survival, clonogenic assays are carried out for all variables considered, with a concentration of 0.5 mg mL This study investigates the enhancement effect of nanoparticles combined with radiotherapy, relating both cell survival and DNA damage to cellular elemental uptake. Different nanoparticles and cancer cells are used, where in all cases, nanoparticles cause a level of enhancement. The complexity in enhancement with increasing uptake can be seen, highlighting different mechanisms contributing to the enhancement effect. Although this form of treatment has shown much promise, it is not currently used clinically due to the number of variables that need to be investigated to control and optimize the effect. Various groups have investigated different aspects, such as varying NP size or radiation beam energy. Even with knowledge from these studies, there is still a considerable amount of variability in reported findings. Differences are caused by the diversity in cell lines, NPs with their respective coatings, incubated NP concentrations, incubation times, irradiation parameters, as well as the assays used to demonstrate the effects. This has led to variations in the results, where significant enhancements of a factor of 25 were shown by Rahman et al., with Aurovist 1.9 nm gold nanoparticles (AuNPs) at a concentration of 1 mm with bovine aortic endothelial cells (BAEC) and 80 kV X‐rays, compared to smaller enhancements shown by Chithrani et al., where they synthesized 50 nm AuNPs at a concentration of approximately 1 nm in HeLa cells and found an enhancement factor of 1.17 with 6 MV X‐rays. As well as this, there are also differences in protocols between research groups, in both maintenance of cells and assays reported.It has been shown over the years that nanoparticles (NPs) can be used to locally enhance the level of dose deposition, In terms of the physical mechanisms, this was attributed to an increase in secondary electrons, where differences in NP size and composition can first lead to differences in interactions with the NPs, but also the number of electrons produced with sufficient energy to carry out further ionizations. Due to these physical mechanisms, it was initially predicted that NP‐enhanced radiotherapy would only be effective with low energy X‐rays due to difference in mass energy absorption coefficients between soft tissue and high‐Z materials, which decreased with increasing incident X‐ray energy. However, following both in vivo and in vitro experiments, significant enhancements were shown with higher energies, demonstrating that other mechanisms were involved in NP‐enhanced radiotherapy. Therefore, other mechanisms were investigated, such as chemical mechanisms. It has been suggested that this involves NPs chemically sensitizing DNA to the damaging effects of radiation, but also increasing radical formations due to the incident radiation activating the surface atoms of NPs. Finally, biological mechanisms were studied, reported as an increase in reactive oxygen species (ROS) formation, oxidative stress, inhibition of DNA repair, and changes to the cell cycle.A review by Her et al. reported on the different mechanisms associated with NP‐enhanced radiotherapy, where the overall effect is a combination of physical, chemical, and biological mechanisms.To further understand the impact of introducing NPs into a cell, two different variables were considered, i) NP material and ii) cell type, all irradiated with 6 MV X‐rays. Focus was directed on these variables, as the relationship between NP material and cell type has not been characterized in terms of enhancement effect related to cellular uptake, where it has been theorized that an increase in cellular uptake would correlate with a higher enhancement effect. Findings from this work were related to the possible mechanisms that may have regulated any observed enhancement. These NPs were used by Hainfeld et al. in the first study demonstrating in vivo NP‐enhanced radiation therapy. Second were spherical gadolinium‐based NPs (GdNPs) , comprising a polysiloxane matrix with cyclic chelates of gadolinium. These NPs have recently completed a phase I clinical trial and are currently in a phase II trial (NANORAD2). Finally, spherical iron oxide NPs (IONPs)  were considered. IONPs have been used in cancer therapy through magnetic hyperthermia, where the NPs produce heat when exposed to a high‐frequency alternating magnetic field. Although IONPs have not been explicitly investigated in NP‐enhanced radiotherapy, they have undergone several in vitro studies for hyperthermia applications, where they have demonstrated cellular uptake. It was therefore of interest to determine if these larger‐sized NPs would demonstrate similar enhancements to the other NPs considered, when combined with radiotherapy. All NP materials have a sufficiently high atomic number to observe the predicted radiosensitization effect. For all NP materials, the same incubation time (24 h) and concentration of 0.5 mg mL−1 was used, such that comparisons could be made. This concentration was shown by Jain et al. to be effective for radiosensitization, when using Aurovist 1.9 nm AuNPs.As the NP material can affect the radiosensitization effect, three commercially available NP types were investigated. The first were spherical AuNPs , with a thiol coating. Other factors such as the surface‐to‐volume ratio can affect uptake, as a larger ratio increases the probability of interacting with cellular receptors for uptake. Differences in uptake across NPs and cell lines were demonstrated by Dos Santos et al., where they investigated the level of uptake in five different cell lines, with negatively charged carboxylated polystyrene (PS–COOH) NPs ranging from 40–500 nm to micrometer‐sized objects (1 and 2 µm). At a concentration of 20 µg mL−1 (24 h incubation), they demonstrated a decrease in cellular uptake with increasing NP size across all cell lines tested. Another aspect is that different cells will respond to radiation differently, where some are more radioresistant, in which case the use of NPs is of particular interest, as it may radiosensitize the cells. The reason for differences in radiosensitivity between cells is not fully understood but thought to be due to factors such as differences in ability to repair damage.The other variable considered was the cell type, which could demonstrate in which cases NPs may be most beneficial. Two cell types were investigated, MCF‐7, a human breast adenocarcinoma cell line, and U87, a human glioblastoma cell line. Both of these cell lines are well characterized and extensively studied within the literature. They demonstrate two different cancer types, where differences in cellular uptake and radiation response were expected; therefore, it allows for trends to be identified across the two cell lines and the different NPs considered. It has been shown in the literature that depending on the cell type, differences in NP uptake can occur due to characteristics of the microenvironment, affecting NPs internalized within the cell. These reports also highlighted that although the α/β ratio would indicate changes in radiosensitivity due to the addition of NPs, no study has currently incorporated this metric within their analysis of the enhancement effect. An interesting aspect of this is to compare findings with the literature on samples irradiated with higher linear energy transfer (LET) radiation, such as protons, which show an increased relative biological effectiveness (RBE) due to more densely ionizing incident radiation. By comparing with in vitro studies that irradiate samples with protons, it is possible to determine if the addition of NPs can alter the α/β ratio of X‐ray irradiations to be comparable to radiation with a higher LET (protons). Finally, the quantification of DNA damage in the form of immunofluorescence staining has been identified as a key factor regulating radiation response, which needs to be quantified in terms of NP‐enhanced radiotherapy. Therefore, this study aims to unify these key metrics in a parameterized study, considering the effect of NP type on cell line, relating cell survival and DNA damage to cellular uptake. This was demonstrated through both clonogenic assays, quantifying cell survival post‐irradiation, and the 53BP1 foci formation assay, which quantifies DNA double‐strand break (DSB) repair through the marker 53BP1. Both results were compared to uptake measurements using inductively coupled plasma mass spectrometry (ICP‐MS). The results from this study offer an understanding of the biological impact of using NPs though an in vitro study, comparing variability in biological effect across both cell line and NP types and relating findings with cellular uptake.These studies have demonstrated the difficulties in assessing the most optimum setup for NP‐enhanced radiotherapy, due to the variability in cell type, NP, and beam characteristics considered. A cooperative of stakeholders reported recommendations to standardize reporting on the efficacy of NP‐enhanced radiotherapy, highlighting metrics such as NP cellular uptake and cell survival (using fitting parameters α and β obtained from the linear‐quadratic (LQ) model), as well as quoting an enhancement ratio at a dose level of 2 Gy.22.1FigureThe effect of a 24 h exposure to NPs was assessed through the clonogenic assay as shown in 2.2FigureUsing ICP‐MS, differences in cellular uptake were reported between cell lines and nanoparticle types following a 24 h incubation period. 2.3FigureTablep = 0.029), 20.5 ± 8.73 Gy (p = 0.047), and 20.5 ± 8.90 Gy (p = 0.049) for GdNPs, AuNPs, and IONPs, respectively, compared to a ratio of 6.02 ± 1.19 Gy for cells alone. For MCF‐7 cells, ratios were 39.6 ± 15.6 Gy (p = 0.021), 39.3 ± 17.4 Gy (p = 0.030), and 22.9 ± 9.91 Gy (p = 0.044) for GdNPs, AuNPs, and IONPs, respectively, compared to a ratio of 6.15 ± 1.32 Gy for cells alone, where p‐values quoted compare samples with NPs to the control without NPs. An increase in the α/β ratio signifies that tumor response is less dependent on the dose per fraction, therefore a lower dose can be used. An interesting point was the significant decrease in β for U87 cells with GdNPs, where a decrease in β indicates a smaller proportion of repairable cell damage. Having shown a significance between NPs and the control , comparisons were made for the same NP between the two cell lines , where no significance was shown for all NPs considered, demonstrating that the NPs had a biological effect independent of the cell line considered.2.4p = 0.008), as shown in Figurep = 0.020) and 1.26 ± 0.07 (p = 0.004) for AuNPs and IONPs, respectively. With MCF‐7 cells, the highest EF was 1.74 ± 0.08 (p < 0.001) with AuNPs, compared to 1.58 ± 0.10 (p = 0.001) and 1.35 ± 0.07 (p = 0.002) for GdNPs and IONPs, respectively. In terms of comparing enhancements between NPs, with U87 cells, there were significant differences between AuNPs and GdNPs (p = 0.026), then GdNPs and IONPs (p = 0.023). With MCF‐7 cells, significant differences were shown between AuNPs and IONPs (p = 0.003), then GdNPs and IONPs (p = 0.034). When comparing the same NP between the two cell lines, a significance in EF was only demonstrated with AuNPs (p* = 0.004).For U87 cells, all NPs considered caused an increase in the level of cell kill, where the highest was with GdNPs showing an EF of 2.02 ± 0.36  was also comparable to those in the literature which ranged from 2 to 10 Gy.Considering the clonogenic assay work, it was evident from the results that the addition of NPs created a significant decrease in cell survival Figure . AlthougWhen comparing the ratios of the same NPs between the two cell lines, no significance was demonstrated for all NPs considered, highlighting that the NPs had an effect on cell kill irrespective of the cell line considered. In terms of enhancement, a statistically significant increase in enhancement was demonstrated with all NPs and cell lines Figure . This coSimilar findings in DNA damage were reported by Taggart et al., where they also considered the use of a breast and glioma cell line . With this study, they only considered one NP type, the same commercial AuNP used in this study; however, they irradiated their samples using 225 kVp X‐rays, rather than the 6 MV linac used in this study. They demonstrated statistically significant increase in DNA damage with the breast cancer cell line; however, with the glioma cell line, they showed a statistically significant decrease in DNA damage. indicating a higher α/β ratio than X‐rays, where a point of interest was to identify if the addition of NPs to cells irradiated by X‐rays could show comparable damage to that observed with higher LET radiation. From the work of Chaudhary et al., the α/β ratio for U87 cells irradiated by protons at an LET of 11.9 keV µm−1 was approximately 10 Gy. Our findings show higher values than this for both types of NPs , indicating a more linear survival curve, similar to that seen with protons, but in our case, even steeper than protons for both NPs. This suggests that the addition of NPs increases the biological effectiveness of the treatment, offering comparable damage to that of higher LET radiation.Another factor for the treatment was the radiation type used, as it is known that when irradiating with higher LET radiation, the survival curve is steeper, which indicates less repaired damage. The linear survival curve with particle radiation is due to an increasing alpha value with increasing LET,−1 (12 h incubation compared to 24 h used in our study), there was a difference in the observed EF, where they report a factor of 1.23, whereas this study found a value of 2.02. A possible reason for this difference is that although both considered GdNPs and the same cell line, incubation times differed. The longer incubation time would have led to different number of NPs internalized within the cell, resulting in a different EF; however, this study did not quote the average amount of Gd taken up within a cell.In comparison to the study by Stefancikova et al., which also used GdNPs, a previous formulation of the commercialized AGuIX NPs, and U87 cells irradiated with X‐rays , at a concentration of 0.5 mg mLFor comparisons to be made between the NP types and cell lines used, quantification of the cellular elemental uptake was needed Figure . It was  thereby reducing both chemical and biological enhancement. Other cellular responses, resulting in biological mechanisms such as oxidative stress and cell cycle effects could have caused differences in the biological effect, which may not be dependent on a higher level of elemental uptake.Interestingly, Figure 4This study was able to demonstrate biological changes encountered by cells due to the presence of NPs combined with ionizing radiation. It was possible to quantify differences between both cell lines and NP types. In terms of the clonogenic assays with U87 cells, an enhancement of dose was observed, whereby a lower dose needed to be delivered (0.98 Gy with GdNPs compared to 2 Gy without NPs) to offer the same level of cell survival as that observed with cells alone at 2 Gy. This study has highlighted the complex relationship between elemental uptake and enhancement effect both in terms of cell survival and DNA damage. It was shown that when considering the same NP between the two cell lines, a higher enhancement effect was related to lower cellular elemental uptake, whereas the DNA damage increased with increasing elemental uptake. This alluded to differences in contribution from mechanisms highlighted in the literature, changing with increasing elemental uptake. Although significance in enhancement was demonstrated by comparing NP‐inoculated to NP‐free samples, significance was only demonstrated for AuNPs when comparing the same NP between the two cell lines. This therefore indicates that the NP type, elemental uptake, or cell type alone cannot explain the radiobiological enhancement effect observed with the NPs and cell lines used in this study. Further work would be to investigate different combinations of these parameters to explain the effects observed. One aspect would be to consider different incubation concentration to demonstrate if an optimum uptake can be identified. Others would be to use specific assays to highlight the different mechanisms in place to demonstrate their overall contribution to cell kill following NP‐enhanced radiotherapy.52/95% air.Two cell lines were investigated, MCF‐7 human breast adenocarcinoma cell line and U87 human glioblastoma cell line. Both cell lines were cultured in Minimum Essential Medium (MEM) , supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin . All cells were maintained in monolayers in a tissue culture incubator at 37 °C with 5% CO−1 of each respective NP material to the seeded cells.Three nanoparticles were investigated; the first were freeze‐dried spherical AuNPs (Aurovist) with a mean diameter of 1.9 ± 0.6 nm  that were re‐dispersed in phosphate buffered saline (PBS)  and stored at 20 °C as per the manufacturer's instructions. The second were freeze‐dried gadolinium‐based nanoparticles (AGuIX) with a mean diameter of 3 ± 1.5 nm . These were re‐dispersed in ultrapure water and stored at 4 °C. The third were magnetic dextran iron oxide nanoparticles (RCL‐01) in water with a mean (Z‐average) diameter of 140 ± 4 nm and a polydispersity index of 0.25 ± 0.02 , stored at 4 °C. All stock solutions were diluted in culture medium before adding a concentration of 0.5 mg mL−1 (24 h incubation) was not toxic; therefore, this and a lower concentration of 0.2 mg mL−1 were tested. Following the incubation period, medium and nanoparticle mixture was removed, wells were gently washed twice with PBS, and each well was filled with 5 mL of fresh medium. The plates were incubated for 10–14 days, fixed, stained, and colonies were counted and compared to control plates without NPs.To ensure the concentrations used were non‐toxic, a fixed number of cells were seeded onto six‐well plates , one plate for each cell line, where two concentrations of each nanoparticle were introduced. From the literature, it was reported that a concentration of 0.5 mg mL−1 was not toxic, a clonogenic assay investigation was conducted in triplicate. For the irradiations, 105 cells mL−1 were seeded onto 35 mm culture dishes  48 h prior to irradiation, to reach between 80–100% confluence on the day of irradiation. The NP samples were incubated for 24 h prior to irradiation with 1 mL medium containing NPs at a concentration of 0.5 mg mL−1. Medium was also changed for the control samples 24 h prior to irradiation. Following the incubation period and just prior to irradiation, NP solution was removed, the samples were gently washed twice with PBS to remove any extracellular NPs, and fresh medium was added to each dish.Having confirmed the concentration of 0.5 mg mL Each sample was then processed, and counts were related to the reference curve to determine the elemental concentration of each material per sample. Results were then reported as the mass of NP element per cell (pg per cell).As with previous samples, cells were seeded, and NPs were added. Following the incubation period, the NP solution was removed, and dishes were gently washed twice with PBS. Cells were trypsinized and counted with a hemocytometer to determine the total number of cells per sample. The cell suspension was centrifuged for 15 min at 1000 RPM. Pellets were then dissolved by aqua regia (three parts hydrochloric acid to one part nitric acid) and the solution was diluted with ultrapure water. Using ICP‐MS , reference measurements were initially carried out on a known concentration of each NP type, diluted to obtain a reference curve relating the counts per second to the NP concentration.−1. Dishes were placed in the center of a 10 × 10 cm2 field  immediately before irradiation. Irradiations were carried out at the National Physical Laboratory, Teddington, UK, with a 6 MV linac (Elekta Versa HDTM), with a dose rate of 6.5 Gy min Following this, colonies were stained with 0.4% crystal violet and counted using a Nikkon Eclipse Ti‐S inverted microscope. The plating efficiency was calculated as the ratio of the colonies formed to cells seeded. The surviving fraction (SF) was calculated as the plating efficiency of the irradiated sample divided by the plating efficiency of the unirradiated sample. The SF was plotted against the dose, where the curves were then fitted to the LQ model described by Equation , using a non‐linear least‐squares fitting procedure, weighted to the errors associated with each measurement. From this the α/β ratio was determined for each curve. The enhancement factor (EF) was determined as the ratio of dose required for NP treated cells to give same survival as cells not treated with NPs, irradiated at 2 Gy, where the ratios were determined using the SF fits . Significance was tested both between samples containing NPs compared to control samples and between the different NP samples.Statistical analysis was performed using a t‐test.For the staining of the DNA damage marker 53BP1, samples were fixed at two timepoints, one 30 min post‐irradiation, another after 24 h, demonstrating the immediate and unrepaired damage post‐irradiation respectively, the latter being referred to as the ‘residual DNA damage' or ‘residual damage' of the samples. Medium was removed and each dish was washed twice with PBS. Cold fixing solution (methanol:acetic acid) was added to each dish and left for 30 min at 4 °C. The solution was then removed and replaced with PBS where samples were stored at 4 °C until immunofluorescence staining. In terms of the staining, samples were washed with cold PBS, permeabilized (0.5% Triton X‐100 in PBS), and blocked in 10% goat serum, 1% BSA, and 0.1% Triton X‐100 in PBS for 1 h at 37 °C in an incubator. Cells were probed with 53BP1 primary antibody raised in rabbit  at a dilution of 1:1000 and incubated at 37 °C for an hour. Samples were washed three times, then probed with goat anti‐rabbit Alexa Flour 488‐conjugatedsecondary antibody  at a dilution of 1:1000 (samples were covered from this point due to light sensitivity), and incubated for an hour. Samples were then washed three times and counterstained with DAPI  where they were then imaged using the CellInsight CX5 High Content Screening Platform . For each data point, foci were manually randomly counted in 50 cells, where each point had triplicate experiments. Statistical significance was determined using a One of the authors (Q.A.P.) is a shareholder in, and part‐time advisor to, companies (Resonant Circuits Limited and Endomagnetics Limited) with commercial interests in magnetic nanoparticles. Another author (O.T.) has to disclose the patents WO2011/135101 and WO2009/053644. These patents protect the AGuIX NPs described in this publication. O.T. is an employee and shareholder of the company NH TherAguix, who are developing the AGuIX NPs.