The protein expression of IKK-α and IKK-β, as inhibitors of NF-KB which exhibit a critical function in the regulation of genes involved in chronic inflammatory disorders, were studied in the tested cancerous cell lines. In this study, the anticancer activity of household-processed fenugreek leaves and seeds against HepG2, HCT-116, MCF-7, and VERO cell lines was measured using an MTT assay. DNA fragmentation of both HepG2 and MCF-7 was investigated by using gel electrophoresis. RT-PCR was used to evaluate the relative expression of each p53, caspase-3, Bax, and Bcl-2 genes, whereas ELISA assay determined the expression of caspase-3, TNF-α, and 8-OHDG genes. Western blotting analyzed the protein-expressing levels of IKK-α and IKK-β proteins in each studied cell line. Data showed that at 500 µg mL−1, ADFL had the highest cytotoxicity against the HepG2 and HCT-116 cell lines. Although, each UFS and GFS sample had a more inhibitory effect on MCF-7 cells than ADFL. Gel electrophoresis demonstrated that the IC50 of each ADFL, UFS, and GFS sample induced DNA fragmentation in HepG2 and MCF-7, contrary to untreated cell lines. Gene expression using RT-PCR showed that IC50 doses of each sample induced apoptosis through the up-regulation of the p53, caspase-3, and Bax genes and the down-regulation of the Bcl-2 gene in each studied cell line. The relative expression of TNF-α, 8-OHDG, and caspase-3 genes of each HepG2 and MCF-7 cell line using ELISA assays demonstrated that ADFL, UFS, and GFS samples reduced the expression of TNF-α and 8-OHDG genes but increased the expression of the caspase-3 gene. Protein-expressing levels of IKK-α and IKK-β proteins in each studied cell line, determined using Western blotting, indicated that household treatments decreased IKK-α expression compared to the UFS sample. Moreover, the ADFL and SFS samples had the most activity in the IKK-β expression levels. Among all studied samples, air-dried fenugreek leaves and unprocessed and germinated fenugreek seeds had the most anti-proliferative and apoptotic-inducing properties against human HepG2, MCF-7, and HCT-116 cell lines, as compared to the VERO cell line. So, these crude extracts can be used in the future for developing new effective natural drugs for the treatment of hepatocellular, breast, and colon carcinomas.Household processing of fenugreek seeds and leaves, including soaking, germination, and boiling of the seeds, and air-drying of the leaves, has improved the levels of human consumption of the bitter seeds and increased the shelf life of fresh leaves, respectively. The potential anticancer activity of either unprocessed or processed fenugreek seeds or leaves and the relative expression of pro-apoptotic and anti-apoptotic genes of the studied cancerous cell lines exposed to IC Cancer remains an aggressive cause of death on a global scale despite significant efforts. The chemotherapeutic agents currently in use have negative side effects and have not performed as expected. Medicinal herbs have been used for ages to cure a variety of illnesses and are also known to have anticancer properties .Tumor chemoprevention is a procedure used to decelerate, degenerate, or prevent the cross-mechanism of tumorigenesis, including blocking its important morphogenetic stages, such as preneoplasia, neoplasia, and metastasis. Alternatives such as naturally occurring phytochemicals found in food are increasingly preferred over manufactured medications for a number of reasons, including less (or no) toxicity or side effects, improved availability, and safety. Programmed cell death (PCD)-type I (apoptosis), PCD-type II (autophagic cell death), and necrosis are some of the mechanisms involved in cell death produced by plant products . A complex biological process called apoptosis is crucial for removing damaged cells from living things. A type of controlled cell death known as apoptosis is characterized by distinctive morphological and biochemical alterations in the cells. The apoptotic cascade is fueled by a variety of proteins. They can be assessed utilizing common analytical procedures based on protein detection. Caspases are enzymes , and their activity can be used to determine the stage and rate of apoptosis, particularly when they are cleaving the peptide bonds of the right substrate. In humans, there are eleven isoenzymes of caspases, seven of which  are involved in apoptosis .The I kappa B protein kinase complex, often known as IKK, is a 700 kDa protein that can phosphorylate the conserved N-terminal serine residues of IκB-α and IκB-β and is triggered by inflammatory signals  . The IKKTrigonella foenum-graecum, is a traditional herbal remedy used for the treatment of diabetes, high cholesterol, wounds, inflammation, digestive problems, etc. It is also thought to have anti-tumor characteristics; however, the exact mechanisms for this activity are still unclear [Fenugreek, or  unclear . Numerou unclear .50 doses of ethanolic crude extracts of each tested sample. Finally, a Western blot assay was used to measure the protein-expressing levels of both IKK-α and IKK-β protein kinases in each cancerous and normal cell line exposed to the IC50 doses of the studied samples.The aim of this work is to evaluate the anti-proliferative activity of air-dried fenugreek leaves (ADFL), unprocessed fenugreek seeds (UFS), germinated fenugreek seeds (GFS), soaked fenugreek seeds (SFS), and boiled fenugreek seeds (BFS) against three cancerous cells from hepatic (HepG2), colon (HCT-116), and breast (MCF-7) cancer cell lines in comparison to normal kidney cell lines (VERO) using MTT assays. AGE, comet assay, RT-PCR, and ELISA methods were used to study the ability of each studied sample on DNA damage and regulation of pro- and anti-apoptotic genes, by exposing p53- caspase-3, Bax, and Bcl-2 of HepG2, HCT-116, MCF-7, and VERO cells to ICAll RPMI 1640 culture media, glutamine, FBS, penicillin, streptomycin, trypsin, primers for p53, caspase-3, Bcl-2, Bax, GAPDH, solvents, dyes, and chemicals were purchased from Sigma-Aldrich, St. Louis, MO, USA. Primary antibodies included TNF-α, 8-OHDG, Cas-3, GAPDH, IKK-α, and IKK-β, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology, . Western blot kits were purchased from Amersham, Buckinghamshire, UK.All cell lines were obtained from the American Type Culture Collection . Anti-proliferative activity was investigated in vitro against three human cancerous cell lines, including the human hepatic (HepG2), breast (MCF-7), and colon (HCT-116) carcinoma cell lines, and normal human kidney cell line (VERO), and determined by the Bioassay—Cell Culture Laboratory, National Research Centre. All cell lines were cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% FBS, and 1% penicillin/streptomycin. Then, sub-confluent cultures (70–80%) were trypsinized (trypsin 0.05%/0.53 mM EDTA) and split depending on the seeding ratio.Trigonella foenum-graecum Linn.) were purchased from the local market in Egypt in the month of December 2019 and identified at the Faculty of Science, Cairo University. The leaves were washed thoroughly with tap water, and the surface water was removed by air-drying under shade for 15 days. Leaves were subsequently dried in a hot air oven at 50 °C for 4 h, homogenized to a fine powder, and then stored at 4 °C.Fresh and healthy leaves of fenugreek ((a)Untreated Fenugreek Seeds (UFS)Two kilograms of fenugreek seeds were purchased from the local market in Egypt, identified at the Faculty of Science, Cairo University, and divided into four groups as follows:Five hundred grams of seeds were cleaned manually to remove dust and foreign particles, crushed into a fine powder flour with the help of Moulinex blender LM 241, and sieved through a 0.5 mm mesh size. The powdered flour was kept at 4 °C to prevent changes until further analysis .(b)Soaked Fenugreek Seeds (SFS)w/v) for 12 h at room temperature. After removing the soaking water, the seeds were air-dried in shade for 5 days, followed by hot air oven-drying at 50 °C for 4 h in a conventional oven, and stored at 4 °C [Five hundred grams of untreated seeds were soaked in tap water at the ratio of 1:5 ((c)Germinated Fenugreek Seeds (GFS)w/v) for 12 h at room temperature. After removing the soaking water, seeds were kept in the dark for germination (tied in a cotton cloth) at 20 °C for 60 h in darkness. After harvesting the sprouts, they were air-dried for 5 days, followed by hot air oven-drying at 50 °C for 4 h in a conventional oven, and stored at 4 °C [Five hundred grams of untreated seeds were soaked in tap water at the ratio of 1:5 ((d)Boiled Fenugreek Seeds (BFS)2O (1:5 w/v). The sample was boiled on a hot plate for 10 min, and then, after the water was discarded, the boiled seeds were air-dried in shade for 5 days, followed by hot air oven-drying at 50 °C for 4 h in a conventional oven, and stored at 4 °C [Five hundred grams of untreated seeds were put in 2 L beaker containing 1250 mL tap H at 4 °C .w/v) 80% ethanol in distilled H2O through sonication for 60 min. Extraction was repeated three times. After filtration, an extract of each sample was condensed until dry (resulting in crude extracts) by use of a rotary evaporator at 40 °C. A known weight of each crude extract was dissolved in appropriate volume of dimethyl sulfoxide (DMSO) and stored at −80 °C until further analysis [One hundred grams of each sample were extracted with 1 L -2,5-diphenyltetrazolium bromide (MTT) assay, according to Mosmann .The qualitative method for assessing cell death by detecting DNA fragments using AGE was performed according to Kasibhatla et al. .50 of each studied sample. The method of Kent et al.  was foll50 doses of the tested samples for 24 h, according to Robert [®cycler software 2.1  to calculate the threshold cycle (Ct) using the second derivative maximum. The fold change value for each gene was determined after normalization to the expression levels of GAPDH as an HK gene, which was calculated using the equation 2−∆∆Ct.Gene expression of pro-apoptotic genes  and Bax, an anti-apoptotic gene, were tested against HepG2, HCT-116, MCF-7, and VERO cells exposed to ICo Robert . Raw dat50 of crude extracts of each studied sample was seeded at a density of 2 × 104/well in a 96-well plate and incubated for 24 h. Absorbance was measured using an ELISA reader . Determination was performed in triplicate, and standard deviation was determined.Quantitative analysis of TNF-α, 8-OHDG, and Cas-3 antibodies using ELISA technique was investigated according to the method of Khalil . Each ceThe Western blot for IKK-α and IKK-β primary antibodies was performed according to the method of Morgia . The exp50 was calculated by using GraphPad prism 7. One-way analysis of variance (ANOVA) was used to analyze the difference between groups by applying the least significant difference (LSD) test with a 5% level of significance (p < 0.05).All assays used in this work were evaluated in triplicate, and the data obtained were represented by the mean ± standard deviation (SD). Statistical Analysis Software (SAS 9.1) was applied for the statistical analysis of data, and IC−1. In contrast, the household processing of fenugreek seeds at 1000 µg mL−1 caused a significant decrease in the cell proliferation of the HepG2 cells, and BFS had the lowest cytotoxicity (56%). The IC50 values were 220 µg mL−1 for ADFL and 320 µg mL−1 for UFS, although these values increased after household treatments. The impacts of household-processed fenugreek leaves and seeds on the cell proliferation of HepG2, HCT-116, MCF-7, and VERO cell lines exposed to serial concentrations of the tested samples are presented in −1 also had the highest anti-proliferative activity (95%) against the HCT-116 cell line, and the germinated (GFS), soaked (SFS), and boiled (BFS) fenugreek seeds reduced the activity compared to the untreated seeds (UFS). The IC50 values were 240 µg mL−1 for ADFL and 275 µg mL−1 for UFS, although these values increased after household treatments. Furthermore, the impact of the tested samples on the human colon cancer cell line (HCT-116) is represented in −1, both the untreated and germinated fenugreek seeds (UFS and GFS) had the highest potential cytotoxicity against the MCF-7 (95%), compared to the other tested samples. ADFL had 92% cytotoxicity at 1000 µg mL−1, SFS had 90% at 2000 µg mL−1, and BFS had 95% at 2000 µg mL−1. The IC50 values of both UFS and GFS were 500 and 620 µg mL−1, respectively. In addition, the soaking and boiling treatments of seeds at 1000 µg mL−1 caused a significant decline  in the cell proliferation of MCF-7. Protodioscin, a steroidal saponin of fenugreek, had an inhibitory impact on the leukemic cell line (HL-60) and a weak inhibitory effect on the growth of the gastric cell line (KATO-III) [On the contrary, as shown in 50 (µg mL−1) values of ADFL, UFS, and GFS were 1660, 1840, and 1940 µg mL−1, respectively. However, the soaking and boiling treatments had no effects on the normal VERO cell line.Finally, the effect of the tested samples on the cell proliferation of the normal kidney cell line (VERO) can be observed in 50 doses of each tested sample can be observed in Agarose gel electrophoresis (AGE) was used to evaluate the DNA fragmentation activity of the tested samples to indicate whether or not the action of the extract is associated with apoptosis. DNA fragmentation of the HepG2 and MCF-7 cancer cell lines treated with the IC50 doses of each studied sample was observed. The treatment of the HepG2 and MCF-7 cell lines with the IC50 doses of the household-processed fenugreek leaves and seeds  caused the fragmentation of DNA in each cancer cell line compared to the untreated control cell lines and the cells exposed to the IC50 of the BFS sample. Therefore, the air-dried fenugreek leaves (ADFL) and unprocessed and germinated fenugreek seeds  induced DNA fragmentation and, thus, apoptosis in both HepG2 and MCF-7 cancer cell lines.As shown in The DNA damage of the HepG2 and MCF-7 cancer cell lines was observed by using single-cell gel electrophoresis (SCGE) (Comet assay), which is able to detect DNA strand breaks (SBs) and oxidative DNA damage in cultured cell lines.50 doses of ADFL, UFS, GFS, SFS, and BFS, which were 13.01, 12.91, 11.41, 10.00, and 9.31%, respectively, for HepG2 cell lines, and were 12.27, 11.10, 9.89, 9.66, and 8.77%, respectively, for MCF-7 cell lines. As presented in 50 doses of the studied samples. The head diameters of the untreated HepG2 and MCF-7 cell lines were 17.61 and 17.15 px, respectively. The values of head diameters are also observable in 50 doses of the tested samples had a decrease in DNA % in the head , except in the BFS sample (84.01%), which was not significantly different compared to the control sample. The values were 76.01, 78.00, 79.00, 81.04, and 81.48% for the MCF-7 cell lines treated with ADFL, UFS, GFS, SFS, and BFS, respectively.The percentages of DNA values in the heads of the untreated HepG2 and MCF-7 cell lines (control) were 84.00 and 81.51%, respectively. In contrast, the HepG2 cell lines exposed to the IC50 of ADFL (4.00 px), UFS (3.94 px), and GFS (3.91 px), but was not significantly changed after treatment with either SFS (3.85) or BFS (3.81) samples. On the other hand, the tail length value of the untreated control cell line of MCF-7 was 3.80 px, and increased to 4.01, 3.98, 3.90, and 3.85 px after exposure to ADFL, UFS, GFS, and SFS, respectively, with the exception of the BFS sample (3.81 px).The data in Furthermore, the percentages of DNA in the head were found to be 22.17% for ADFL, 21.00% for UFS, 19.41% for GFS, 18.12% for SFS, and, lastly, 18.00% for BFS relative to the untreated control HepG2 cell lines. In addition, the untreated MCF-7 cell line (control) had a DNA% in the head of 18.20%, which was also raised to 23.21, 22.11, 20.00, and 18.41% for ADFL, UFS, GFS, and SFS, respectively, but did not vary with the BFS sample (18.21%). 50 of ADFL (1.10), UFS (1.07), GFS (1.01), and SFS (0.96), but not with the BFS sample (0.94). Lastly, the tail moment values of DNA of the untreated (control) HepG2 and MCF-7 cell lines were 1.02 and 0.93, respectively. These values were higher after the exposure of HepG2 cell lines to ADFL (1.16), UFS (1.14), GFS (1.10), and SFS (1.07), but did not differ from the BFS sample (1.01). The untreated control MCF-7 cell lines had a tail moment of 0.93 and increased after treatment with the IC50 doses of the selected sample, is summarized in The gene expression of the pro-apoptotic genes involved either p53, Caspase-3 (Cas-3), or Bax, as well as the anti-apoptotic Bcl-2 gene, against HepG2, HCT-116, MCF-7, and VERO cell lines treated with IC50 doses of ADFL, UFS, GFS, SFS, and BFS are compared to the untreated cell lines (control). The results showed that air-dried fenugreek leaves (ADFL) were the most potent sample, which significantly up-regulated p53, Cas-3, and Bax  and down-regulated Bcl-2 (at a 0.52 fold change) compared to untreated cell lines. However, the standard drug (doxorubicin) caused an up-regulation of the studied pro-apoptotic genes, as follows: 1.87 for p53, 3.82 for Cas-3, and 3.11 fold change for Bax genes, and a down-regulation of the Bcl-2 gene (at a 0.32 fold change). In addition, each of the UFS, GFS, SFS, and BFS samples significantly increased the up-regulation of p53 , Cas-3 , and Bax  genes relative to the untreated control cell lines. According to 50 doses of ADFL, UFS, GFS, SFS, and BFS compared to both the control cell line and the other samples that were treated with doxorubicin were also used to study the apoptosis-inducing property of the tested samples  exposed to IC samples B. Data s50 in each studied sample were compared to the untreated cell lines, and the other samples treated with doxorubicin were also used to evaluate the apoptotic-inducing activity of the tested samples  treated with IC samples C. Findin50 concentration of each studied sample was also used to study the apoptosis-inducing properties of the tested samples  exposed to the IC samples D. The da50 doses of household-processed fenugreek leaves and seeds involved ADFL, UFS, GFS, SFS, and BFS, which enhanced the up-regulation of the studied pro-apoptotic p53, Cas-3, and Bax genes and down-regulation of anti-apoptotic Bcl-2 genes, which induced apoptosis of the studied cell lines, then decreased the tumor growth and cell proliferation.According to these findings, we summarized that the treatment of hepatic, colon, and breast cancer cell lines exposed to IC−1 protein, respectively, for each HepG2 and MCF-7 cell lines treated with IC50 doses of ADFL, UFS, GFS, SFS, and BFS. This caused a significant decline up to 51 and 57%, 61 and 61%, 72 and 81%, 89 and 92%, and finally, 96 and 99%, respectively for the same tested samples. The control (untreated cell line) showed 25.03 and 23.00 pg mg−1 protein for each untreated HepG2 and MCF-7 cell line.Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine which is involved in immune function and is also proposed to play a role in metabolic disorders. The results presented in An 8-Hydroxy-2′-deoxyguanosine (8-OHDG) biomarker was evaluated to measure DNA damage. The results presented in The caspase-3 (Cas-3) factor is usually used to evaluate the apoptotic cascade. This biomarker factor was activated and increased after exposure to the HepG2 and MCF-7 cell lines to the studied samples, with the exception of the boiled fenugreek seeds (BFS). According to the presented data in To further confirm the results of the IKK-α and IKK-β expressions, the signaling pathway was further investigated through a Western blotting assay . 50 doses . Meanwhile, IKK-α expression levels in VERO cells were not detected in all studied samples, except for UFS (60.78%) and GFS (64.83%).As observable in 50 doses . Meanwhile, IKK-β expression levels were been high in HCT-116 cells treated with SFS (46.90%), GFS (45.57%), and ADFL (44.56%), respectively.In contrast, the results shown in The fenugreek plant has had an important role for a long time in the treatment and prevention of diseases. Various studies have also proved many of these conventional applications and have observed the beneficial value of this plant and its capabilities in traditional medicine. However, the scientific information about the evaluation of the mechanism of action of this plant is not enough .Finding naturally occurring chemo-preventive compounds has received more attention in recent years, especially those found in dietary and medicinal plants due to their bioactive components ,23. These findings are in agreement with Raju  and ThirThe majority of these bioactive chemicals inhibit cell cycle progression and cause apoptotic cell death to carry out their cancer-chemotherapeutic activities. As a result, the induction of apoptosis in tumor cells has evolved into a marker of the tumor treatment response when using a bioactive chemical derived from plants to lower and manage cancer-related mortality in humans ,27. TherIn this work, household processes were been involved in the air-drying of fenugreek leaves for preserving the leaves from spoilage resulting from the moisture content of fresh leaves, and soaking, germination, and boiling of raw fenugreek seeds to reduce the bitter taste, due to the presence of saponins, and specific smell, due to the presence of alkaloids and volatile oils, which limit their usability and acceptability in food industries. This study aimed to evaluate the anti-proliferative activity of air-dried fenugreek leaves (ADFL), unprocessed fenugreek seeds (UFS), germinated fenugreek seeds (GFS), soaked fenugreek seeds (SFS), and boiled fenugreek seeds (BFS) against three cancerous cells involved hepatic (HepG2), colon (HCT-116), and breast (MCF-7) cells in comparison to normal kidney cell lines by using an MTT assay.A typical genetic change linked to cancer is the mutation of the tumor suppressor p53, which inactivates 50% of human malignancies . A complDNA damage is one of the most well-known effects of oxidative stress. Biomarkers for nucleic acid oxidation are frequently utilized to evaluate this damage . The mosThrough the inhibition of Akt activity, diosgenin reduced TNF-induced NF-κB activation, IκB-α kinase activation, IκB-α phosphorylation, IκB degradation, p65 (RELA) phosphorylation, and p65 nuclear translocation. Diosgenin also inhibited the TNF-induced production of genes related to cell proliferation , anti-apoptosis , and invasion -9). Additionally, TNF and chemotherapeutic medicines such as paclitaxel and doxorubicin that promote apoptosis were potentiated by diosgenin .The primary public health issue across the globe is cancer. The identification of naturally occurring chemo-preventive compounds has recently received increased interest, particularly those found in dietary and medicinal plants because of their bioactive components. The majority of these bioactive chemicals inhibit cell cycle progression and cause apoptotic cell death to carry out their cancer-chemotherapeutic activities. As a result, the induction of apoptosis in tumor cells has evolved into a marker of the tumor treatment response when using a bioactive substance produced from plants to lower and manage cancer-related mortality in humans. When compared to the VERO cell line, air-dried fenugreek leaves, raw fenugreek seeds, and germinated fenugreek seeds had the most anti-proliferative and apoptosis-inducing effects on each of the human HepG2, MCF-7, and HCT-116 cell lines. So, these crude extracts can be used in the future for developing new effective natural drugs for the treatment of hepatocellular, breast, and colon carcinomas.


The single crystal X-ray analysis of the 2a complex revealed that a mononuclear and a dinuclear complex co-crystallize in the solid state. The electronic structures of the complexes are optimized by Density Functional Theory (DFT) calculations. The monomeric nature of 2a and 2b species is maintained in solution. Antioxidant activities of the ligands  and Cu(II) complexes  were determined by in vitro assays such as 1,1-diphenyl-2-picrylhydrazyl free radicals (DPPH.) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radicals (ABTS+). Our results demonstrated that 2a showed better antioxidant activity. MTT assays were performed to assess the toxicity of ligands and Cu(II) complexes in V79 cells. The antiproliferative activity of compounds was tested against two human tumor cell lines: MCF-7 (breast adenocarcinoma) and SW620  and on MRC-5 . All compounds showed high cytotoxicity in the all-cell lines but showed no selectivity for tumor cell lines. Antiproliferative activity by clonogenic assay 2b showed a more significant inhibitory effect on the MCF-7 cell lines than on MRC-5. DNA damage for the 2b compound at 10 µM concentration was about three times higher in MCF-7 cells than in MRC-5 cells.Cu(II) complexes bearing NNO-donor Schiff base ligands ( Metallodrugs are compounds that have promising prospects for meeting biomedical challenges. These pharmaceuticals have been applied successfully in antineoplastic chemotherapy for different cancers ,5,6,7,8.Copper is among the most examined metal ions due to its significant role in cancer ,18. AbnoSchiff bases containing azomethine group (–CH=N–) are scaffolds of high interest in medicinal chemistry due to their interesting pharmacological properties, thermal stability, high synthesis, flexibility, and chelating ability with metal ions ,36,37,38Cells in tumors present higher levels of ROS and are particularly sensitive to oxidative stress. This way, antioxidants are being explored to find more effective cancer therapies. Antioxidants can minimize the burden of free reactive radicals in cells caused by chemotherapeutic agents . So, it Based on these facts, we are interested in determining the impact of amine moiety changes (both electronic and rigid vs. flexible) on the biological activity of the two copper complexes containing Schiff base chromophore and phenoxide fragment at the second and third coordination positions, respectively.9H7N. This compound shows weak tertiary base properties because the nitrogen atom in the structure pulls electrons by resonance  (1a), and  (1b) ligands were prepared according to the procedure described in + . UV-Vis : 229, 266, 290, 348, 647. UV-Vis : 279, 312, 382, 675.Synthesis of copper complex 2b: This complex was prepared as described above for 2a, starting from 1b  and CuCl2·2H2O  to give 2b as a brown solid. Yield: 87%. Anal. Calcd. for C24H27ClCuN2O (2a): C 62.87; H 5.94; N 6.11. Found: C 61.38; H 6.23; N 5.95. ESI-HRMS  = 422.1416 [M-Cl]+ . UV-Vis : 237, 288, 348, 483. UV-Vis : 260, 346, 472.Synthesis of copper complex A Bruker D8 Venture Photon 100 dual source diffractometer was used to collect X-ray data for structural analysis. Data were collected using Mo-Kα radiation and a combination of ϕ and ω scans was carried out to obtain at least one unique data set. The crystal structures were solved using direct methods with SHELXS . The fin1a, b and its Cu(II) complexes 2a, b were performed using the ORCA program package  were obtained from the Rio de Janeiro Cell Bank . The cell lines were grown in DMEM supplemented with 10% heat-inactivated FBS or RPMI-1640 supplemented with 20% FBS, 0.2 mg/mL L-glutamine, 100 IU/mL penicillin, and 100 µg/mL streptomycin. Cells were kept in tissue culture flasks at 37 °C in a humidified atmosphere containing 5% CO4 cells/well) in complete media. V79 cells were treated for 24 h with all compounds at concentrations of 1, 10, 50 and100 µM. For human cells, concentrations of 1, 10, 20, 30, 40, 50, 75, 85, and 100 µM of the ligands  and Cu(II) complex 2a for 24 h and 72 h were applied. Treatment with complex 2b evaluated concentrations of 1, 2, 4, 10, 20, and 30 μM for 24 h and 1, 2, 3, 4, 5, 7.5, 8.5, and 10 μM for 72 h in all human cell lines. All compounds were dissolved in DMSO and added to the medium to reach the desired concentrations (0.1% of DMSO). The activity of this solvent alone was measured by a blank sample solution containing the same amount of DMSO. After the exposure period, the culture media were replaced with the addition of 100 µL serum-free media containing the 3--2,5-diphenyltetrazolium bromide) (MTT) dye (1 mg/mL), and incubated for 3 h at 37 °C After incubation, the supernatant was removed, the residual purple formazan product was solubilized in 150 µL DMSO, and its absorbance was measured at 570 nm. The assay was performed in triplicate. Cell viability (%) and IC50 (drug concentration required to inhibit the cell growth by 50% after 24 h or 72 h of incubation) were obtained through GraphPad Prism 5 . The cytotoxic potential of all compounds was measured by the methylthiazol tetrazolium (MTT) assay in Chinese hamster lung fibroblasts and human cell lines. Cell lines were cultured in a 96-well plate  and Cu(II) complexes 2a  and 2b (1 and 4 μM) for 72 h. The cells were cultured in a complete medium without compounds 2a and 2b (control) for another 8 days for the MRC-5 strain, 10 days for SW620, and 14 days for MCF-7. The cell culture medium was changed to a new medium every 2 days. Cells were washed twice with PBS and fixed with methanol for 30 min, followed by staining with 0.5% crystal violet. The assay was performed in triplicate. Colonies were manually counted, and colony survival was presented as a percentage relative to the respective negative control.MCF-7, MRC-5, and SW620 human cell lines were seeded in 6-well plates at a density of 300 cells per well containing 1 mL of medium. After 24 h of incubation, cells were treated with the ligands 4 cells were seeded on twenty-four-well plates and incubated for 24 h. Cells were incubated with various concentrations of 1a, b and 2a, b for 3 h in an FBS-free medium. Each dose was evaluated by three independent experiments. The alkaline comet assay was performed as described by Singh et al. + ions for 2a (m/z = 414.1711), and [M–Cl]+ ions for 2b (m/z = 422.1416) , and one absorption band in the region of 647–672 nm associated with d–d transitions.The ESI-HRMS results indicated that the monomeric nature of 22.1416) . UV–visi22.1416)  75]. In. In2a an2a complex was established by X-ray diffraction studies of a single crystal. The solid-state structure of 2a is shown in 2a) and a dinuclear (2a’) complexes of 2a co-crystallize in the solid state. In solution, there are only mononuclear species, according to the ESI-MS studies  Å and 2.8452(6) Å, and the Cu–Cl–Cu bond angle is 89.481(17)°. The Cu–Cl bond of 2.8452(6) Å was longer than the sum of the covalent radii (2.31 Å), indicating the existence of weak interactions between the chloride and the copper center .The Cu⋯Cu distance is 3.6111(4) Å. Otherwise, the mononuclear complex has only a short intermolecular contact through hydrogen bond see , with Cu2a, 2a’, and 2b. All optimized structures for the ground state obtained at the B3LYP/def2-TZVPP/def2-SVP level of theory in the gas phase are shown in Considering the X-ray diffraction analysis results that showed the presence of mono- and dinuclear species in the solid state, we decided to carry out DFT calculations to obtain further insights about the minimum energy structures of 2a and 2b exhibited a distorted quadratic structure, with Cu–Cl bond distances within 2.219 and 2.235 Å, Cu–Nimine 1.937 and 2.000 Å, Cu–Ophenoxy 1.954 and 1.923 Å, and Cu–N with a distance of 2.109 and 1.962 Å. The dinuclear complex (2a’) has a slightly distorted square pyramid geometry with chloride bridge ligands . Thus, the distance between the copper atom and the bridging Cl ligand obtained experimentally (2.8 Å) and by DFT calculations (2.9 Å) strongly suggest a weak electrostatic interaction between two monomeric structures that affords a more stable dimeric species.DFT calculations showed that the energy difference (ΔHOMO-LUMO). The HOMO (EHOMO) and LUMO (ELUMO) energies for the ligands and complexes provide information about energy distribution and energetic behavior. The negative magnitude of EHOMO and ELUMO establishes the stability of compounds  and a isplatin )], respe50 value of the compounds between the normal and malignant cell lines, it is noted that the compounds do not show selectivity for tumor cells (MCF-7 and SW620) in this assay. When comparing the IC1a, b) and Cu(II) complexes . The clonogenic assay is based on the ability of a single cell to grow in a colony, being more reliable in evaluating the selectivity of each compound in cell proliferation. Inhibition of cell proliferation can occur by inducing death , by mitotic catastrophe, or even by senescence [Clonogenic assay was used to confirm the inhibition activity towards human cell lines MCF-7, SW620, and MRC-5 of the ligands  and double-strand breaks (DSBs), alkali-labile sites, DNA adducts, excision repair sites, and cross-links). As can be seen in 1a compound increases damage index (DI) at concentrations of 10–100 µM in MRC-5 and MCF-7 cell lines, as well as 50 µM and 100 µM in the SW620 cell line. The compounds 2a and 1b increase DI at 50 µM and 100 µM in MRC-5 and MCF-7 cell lines, as well as ≥10 µM in the SW620 cell line. The 2b compound increased DI in low concentrations, at concentrations ≥4 µM in MCF-7 and SW620 cell lines. In fact, DI for 2b compound at 10 µM concentration was about three times higher in MCF-7 cells than in MRC-5 cells. Further studies with 1a, b and 2a, b compounds should be performed to provide a better understanding of the mechanisms underlying the cytotoxic and genotoxic effects.Considering the cytotoxic effects observed we investigated if the 2+ is a redox-active metal ion and can cause oxidative DNA damage and strand breaks. Furthermore, Cu2+ ions are capable of binding coordinatively with nuclear proteins and DNA causing site-specific damage [All compounds were able to induce DNA damage. Cuc damage . This rec damage .2a and 2b were performed to analyze if these compounds are DNA-targeting agents. Results from docking simulations show that the compounds have similar affinity values for DNA, of 6.9 and 7.4 kcal mol−1 for 2a and 2b, respectively. Both complexes bind to the minor groove of DNA , where the compound is bonded to the major groove of DNA  complex with the Cu-Cl bond distance indicating weak interaction between the chloride and the copper center. In both cases, the copper centers are chelated by one K3-O,N,N-phenoxy-imine-amine ligand. The copper center is tetracoordinated with a distorted quadratic geometry in the mononuclear species. DFT calculations showed that the energy difference (ΔE) between the dinuclear and mononuclear complexes is only −7.42 kcal.mol−1. Furthermore, DFT calculations established the optimized structure of ligands and their corresponding Cu(II) complexes. The findings revealed a good correlation between experimental and theoretical data. The ESI-HRMS results indicated that the monomeric nature of 2a and 2b species is maintained in solution. The electrochemical behavior of ligands and their Cu(II) complexes were studied.In this study, two new copper complexes chelated by an amine moiety N-donor, Schiff base chromophore, and phenoxide fragments have been developed. Single crystal X-ray studies for 2a has shown better antioxidant activity. All compounds had cytotoxicity and genotoxic effects under tissue culture conditions, and complex 2b induced about three times higher genotoxicity in MCF-7 cells than in MRC-5 cells at a concentration of 10 µM. Thus, low 2b doses may be useful in the development of adjuvant therapies or rational combinations that may be predicted to have synergistic or additive effects in combination with currently used chemotherapeutics. Our in silico analysis sheds light at the molecular level on potential 2a, b-modulated interactions with DNA. These compounds exhibit a high degree of interaction with DNA due to the presence of an extended aromatic ring in 2b. In addition, they also have an amine charged with a hydrogen donor in 2a, demonstrating that these compounds have great potential for biomedical applications.Our study demonstrated that complex


To characterize the cellular responses of murine and human mesothelioma cell lines to different doses of photon radiation with a long-term aim of optimizing a clinically relevant in vivo model in which to study the interaction of radiation therapy and immunotherapy combinations.Two murine mesothelioma cell lines (AB1 and AE17) and 3 human cell lines  were used in the study. Cells were treated with increasing doses of photon radiation. DNA damage, DNA repair, cell proliferation, and apoptosis at different time points after irradiation were quantified by flow cytometry, and cell survival probability was examined using clonogenic survival assay.DNA damage increased with escalating dose in all cell lines. Evident G2/M arrest and reduced cell proliferation were observed after irradiation with 8 Gy. DNA repair was uniformly less efficient at higher compared with lower radiation-fraction doses. The apoptosis dose response varied between cell lines, with greater apoptosis observed at 16 Gy with human BYE and murine AB1 cell lines but less for other studied cell lines, regardless of dose and time. The α/β ratio from the cell survival fraction of human mesothelioma cell lines was smaller than from murine ones, suggesting human cell lines in our study were more sensitive to a change of dose per fraction than were murine mesothelioma cell lines. However, in all studied cell lines, colony formation was completely inhibited at 8 Gy.A threshold dose of 8 Gy appeared to be appropriate for hypofractionated radiation therapy. However, the radiation therapy doses between 4 and 8 Gy remain to be systematically analyzed. These observations provide an accurate picture of the in vitro response of mesothelioma cell lines to photon irradiation and characterize the heterogeneity between human and murine cell lines. This information may guide in vivo experiments and the strengths and limitations of extrapolation from murine experimentation to potential human translation. Malignant pleural mesothelioma (MPM) is an aggressive cancer of the pleura, predominantly arising from asbestos exposure.9+),,KRAS-mutant lung cancer,As demonstrated in preclinical models in other cancer types,P = .001), and Ipi-RT had a significantly higher complete response rate than ipilimumab did alone (25% vs 6.5%). Several other studies have reported an abscopal response in patients with advanced melanoma after RT plus ipilimumab,30In a recent phase 2 clinical trial in early-stage non-small cell lung cancer, 60 patients were randomly assigned to receive either durvalumab alone or durvalumab plus stereotactic body radiation therapy (8 Gy × 3); pathologic responses were 53.3%  versus 6.7% , respectively, and several complete pathologic responses were observed in the combination group.+ T-cell phenotype observed after the addition of anti-CTLA-4. Delayed tumor growth in the secondary tumor after irradiation to the primary tumor compared with the sham-RT group was also observed. The authors also reported increased expression of genes such as interferon-γ, granzyme B, ICOS, CD80, and CD86 after RT plus anti-CTLA-4.For mesothelioma, however, the benefits of rRT and immunotherapy are currently understudied in both the clinical and preclinical settings. To date, 1 mouse study using the AB12 mesothelioma model to examine the effects of radioimmunotherapy demonstrated an increased T-cell influx into primary and secondary tumors after hypofractionated RT, with a highly activated CD8,Owing to the paucity of data from the clinic in combining RT and immunotherapy in mesothelioma, it is important to study this approach in a robust animal model.,The effects of RT are exerted primarily through damage to cellular DNA.,Murine mesothelioma cell lines (AB1 and AE17) were generated as previously described.6 cells/mL) at room temperature, using a Gammacell irradiator 3000 SN 211  at various doses as specified. The dose rate from the cesium-137 source was 3.87 Gy/min. The unit was calibrated according to National Institute of Standards and Technology standards.Exponentially growing mesothelioma cell lines were irradiated in 50-mL tubes , 3 minutes) to remove the irradiated media immediately after radiation. Supernatants were discarded, and cell pellets were resuspended in fresh RPMI medium; cells (0.5 × 106) were transferred to a T175 flask and incubated at 37°C in a humidified 5% CO2 atmosphere. BrdU (20 µg) was added to the appropriate flask for the last hour of incubation, and cells were harvested at either 1, 6, 24, 48, or 72 hours after radiation exposure.Assessment of cell proliferation after photon radiation exposure was done by pulse labeling the irradiated cells with 5-bromo-2′-deoxyuridine (BrdU), followed by quantification of the proportion of BrdU2 atmosphere. The cells were washed by resuspension in 10 mL culture media to deactivate trypsin and centrifuged . Supernatants were discarded, and cell pellets were resuspended in 1 mL culture media. Cell counts were performed using trypan blue exclusion; 0.5 × 106 cells were collected and washed twice with 2 mL 1X PBS and centrifuged . Supernatants were discarded, and cell pellets were fixed with 100 µl Cytofix/Cytoperm Fixation and Permeabilization solution  for 20 minutes on ice. After cell fixation, cells were washed with staining buffer , transferred into 1 mL freezing medium , and stored at –80°C until staining.One hour after exposure, BrdU pulse labeling was completed at each time point. Cell culture medium was transferred from flasks to 50-mL tubes. The flasks were washed with 10 mL 1X phosphate-buffered saline (PBS), and 2 mL warm trypsin was added to dislodge adherent cells, which were incubated for 2 minutes at 37°C in a humidified 5% CO2 atmosphere. After deoxyribonuclease treatment, the cells were washed with 500 µL 1X PermWash. Supernatants were discarded, and cell pellets were stained using 20 µL antibody cocktail—mouse anti-BrdU-PerCP-C5.5 , mouse anti-γ-H2AX-AF467 , and mouse anticleaved PARP-PE —diluted in staining buffer, then incubated in the dark for 20 minutes at room temperature. After staining, cells were washed with 500 µL 1X PermWash by centrifugation . Cell pellets were then stained with DAPI  for 15 minutes. After staining, cells were washed with 1 mL staining buffer and resuspended in 200 µL staining buffer. All staining tubes were acquired on BD LSRFortessa (BD Biosciences), with 100,000 events recorded per sample. Data were analyzed using Flowjo_V10 .Flow cytometry staining was performed using “apoptosis, DNA damage, and cell proliferation” kits  according to the manufacturer's instructions. In brief, cells frozen at –80°C were thawed at room temperature and transferred to corresponding labeled staining tubes. Cells were washed twice with 2 mL staining buffer and centrifuged . Supernatants were discarded, and cell pellets were fixed with 100 µL BD Cytofix/Cytoperm Fixation and Permeabilization solution for 20 minutes on ice. After fixation, cells were washed with 500 µL 1X PermWash from the BD Cytofix/Cytoperm kit by centrifugation . Supernatants were discarded, and cell pellets were treated with 100 µL deoxyribonuclease (300 µg/mL) in sterile 1X PBS to expose BrdU epitopes, then incubated for 1 hour at 37°C in a humidified 5% CO2 atmosphere, and the media were changed every 3 days. Fifteen days after irradiation, cells were fixed with 100% methanol for 5 minutes and stained with 0.01% crystal violet. Colonies comprising 50 cells or more were counted using ColCount . According to the linear quadratic model, the survival fraction (SF) is mathematically related to dose D, using the relationshipExponentially growing cells were irradiated (1-8 Gy) in 50-mL tubes, then seeded into T25 flasks in triplicate. Flasks were incubated at 37°C in a humidified 5% COb (slope), c (lower limit), d  are fitting parameters.diBrdU is the outcome at dose d  on the ith cell  and depends on the β parameters in linear fashion; α, β1, β2, and β3 represent the fixed intercept ; and εi is the overall error term in the model. Data are presented as the mean ±1 standard deviation unless otherwise stated, and the experiment was independently repeated at least 3 times.Data were analyzed and visualized using the R environment for statistical computing (version 4.0.5). Multiple group comparisons were performed using 1-way analysis of variance with Tukey adjustment. The analysis of covariance was used to compare group means in a multiple linear regression model for DNA repair with Tukey adjustment. A 4-parameter log-logistic regression model for DNA damage dose response was fitted through the relationshipP = .01; P = .01; We first examined DNA damage response using the level of γ-H2AX at 1 hour after radiation. The flow cytometry gating strategy for γ-H2AX is demonstrated in supplementary Fig. E1. The level of γ-H2AX increased as the radiation dose increased, reaching a maximum at approximately 8 Gy for the AB1, AE17, BYE, and JU cell lines A-D. InteP = .008; DNA damage induces cell-cycle arrest at specific checkpoints for cells to repair DNA.P = .01; A temporary cell-cycle arrest was also observed in the AE17 cell line after 8-Gy irradiation, because the level of G2/M was significantly higher than in untreated cells at 24 hours P = .01; B. AdditiWe also observed a decreased proportion of the G0/G1 population in the BYE and JU cell lines at 72 hours at 8 and 16 Gy compared with 0 Gy C-D. Howe,P = .01; Rapid reduction in γ-H2AX expression over time after irradiation suggests efficient DNA repair.We observed no change in the pattern of cell proliferation, compared with unirradiated cells, in any of our cell lines at any postirradiation time point after treatment with 1, 2, or 4 Gy  polymerase (PARP-1) enzyme by flow cytometry . Dose responses varied widely between cell lines; the human BYE and murine AB1 lines demonstrated approximately 10% of cells undergoing apoptosis 24 hours after a radiation dose of 16 Gy (increasing to 20% in the case of BYE after a further 24 hours) A-C. HoweRT is widely used to treat patients with mesothelioma.,As would be expected, we found that γ-H2AX increased with radiation dose in our studied cell lines, confirming a dose-response relationship. Maximum damage was induced by 8 Gy, above which γ-H2AX generation saturated. This finding was consistent with previous studies examining the phosphorylated histone H2AX by flow cytometry after exposure to ionizing radiation in human endothelial cells,,,,,Again, as expected, we showed that higher-dose radiation arrested cells at the G2/M phase, consistent with previous studies demonstrating G2/M arrest by radiation in leukemia and glioblastoma cell lines., it is still difficult to conclude whether this phenomenon will occur in in vivo, or indeed, in the clinic, where fractionated RT is normally used; further research is therefore warranted. Moreover, by fitting multiple doses in a joint model of all cell lines, DNA repair was similar in all the studied cell lines, suggesting that this repair pattern occurs in both animal and human cells.We also characterized DNA repair kinetics by modeling the decrease in γ-H2AX over time after photon irradiation. Gamma-H2AX is a DNA repair protein attracted to sites of DNA damage as the cells are arresting, thereby providing sufficient time for cellular repair.,,The most important measurable outcomes after radiation treatment in our study were cell proliferation, cell death (apoptosis), and cell survival as assessed by clonogenic survival assay. Our finding demonstrated that although lower-dose radiation (1-4 Gy) did not inhibit cell proliferation, high doses (16-32 Gy) reduced proliferation almost completely from 24 hours in murine cells, findings similar to those from Yao et al,,We found the AB1 and BYE human mesothelioma cell lines displayed more apoptosis between 8 Gy, 16 Gy, and 32 Gy, with a time dependence. Our finding is consistent with previous studies demonstrating the apoptotic sensitivity of colorectal cancer lines and human lung cancer.In conclusion, in murine and human mesothelioma cell lines, radiation was shown to induce DNA damage and G2/M arrest, inhibit cell proliferation, and cause apoptosis in a defined and quantifiable manner. Compared with lower doses (1-2 Gy), a dose of 8 Gy demonstrated significantly increased damaging effects. Considering DNA damage repair and the apoptotic mechanism of cells at the cellular level after different doses of irradiation, it can be concluded that an approximate dose of 8 Gy may be an optimal hypofractionated dose to induce cellular damages in our studied cell lines and may be able to generate immunogenic cell death to activate local and systemic immune response for future radioimmunotherapy study in a mouse model. However, because only epithelioid human mesothelioma cell lines were studied, our observation does not necessarily apply for other nonepithelioid mesothelioma, which are typically more resistant to radiation. Additionally, we have identified a 72-hour postradiation time point at which increased inhibition of cell proliferation was consistently observed across several cell lines; this may be an ideal time for combination with immunotherapies , because response rates in mesothelioma are still generally low. Cytotoxic chemotherapy has been shown to improve response rates to anti-PD-L1 in mesotheliomaGiven that the recent approval of nivolumab plus ipilimumab as first-line treatment for mesothelioma significantly improved progression-free survival only in patients with nonepithelioid cancer,


Protein expression profiles are directly related to the different properties of cells and are conditioned by the cellular niche. As an example, they are the cause of the characteristic cell plasticity, epithelium–mesenchymal transition (EMT), and drug resistance of cancer cells. This article characterizes ten biomarkers related to these features in three human colorectal cancer cell lines: SW-480, SW-620, and DLD-1, evaluated by flow cytometry; and in turn, resistance to oxaliplatin is studied through dose–response trials. The main biomarkers present in the three studied lines correspond to EpCAM, CD-133, and AC-133, with the latter two in low proportions in the DLD-1 line. The biomarker CD166 is present in greater amounts in SW-620 and DLD-1 compared to SW-480. Finally, DLD-1 shows high values of Trop2, which may explain the aggressiveness and resistance of these cells to oxaliplatin treatments, as EpCAM is also highly expressed. Exposure to oxaliplatin slows cell growth but also helps generate resistance to the treatment. In conclusion, the response of the cell lines is variable, due to their genetic variability, which will condition protein expression and cell growth. Further analyses in this area will provide important information for better understanding of patients’ cellular response and how to prevent resistance. Colorectal cancer (CRC) is the third most frequent malignant tumor worldwide (1.8 million cases per year). Moreover, it is the cancer with the third highest mortality rate, representing 8.9% of deaths due to cancer ,3. FortyDespite increased incidence in recent years, mortality has decreased, probably due to early detection strategies . The cur2, and it is administered over a period of 2 h, repeated at two-week intervals. If neurotoxicity is persistent, the dose is reduced to 65 mg/m2. Moreover, oxaliplatin should always be administered before fluoropyrimidines [Oxaliplatin is a platinum compound that binds to plasma proteins and distributes throughout all body tissues, inhibiting the synthesis of DNA in the cells due to crosslinks at susceptible sites in the DNA . The useimidines . Irinoteimidines . The resimidines  describeAlthough many patients are free of disease after treatment of the primary tumor, around 50% relapse within the first five years after tumor extraction . This isABCG2), CD133 (PROM1), CD34 and EpCAM [TACSTD2), AC133, CD36, CD166 (ALCAM), variants of CD44 (CD44v6), etc. [Some biomarkers that were first described for this purpose are BCRP1 , etc. ,23,24,256), etc. ,27, wher6), etc. ,29. CD346), etc. ,31. For 6), etc. ,33,34 anIn this study, we employed three human colorectal cancer cell lines with different metastatic potential: SW-480, SW-620 and DLD-1. The SW-480 cell line is an epithelial cell line from the primary tumor of a human colon adenocarcinoma. The SW-620 cell line was isolated from a metastatic carcinoma from the same patient. Finally, DLD-1 is a cell line that proceeds from a human colon adenocarcinoma in Duke’s C stage. The three cell lines are characterized by the expression of the carcinogenic antigen CEA and p53 mutations. However, positive expression of some oncogenes such as KRAS, myc, myb, fos and sis has been detected in the DLD-1 and SW-620 cell lines. Conversely, SW-480 is the only one that conserves the normal expression of nuclear beta-catenin and the sensitivity to hormonal treatment that proceeds from a human colon adenocarcinoma in Duke´s C stage.The aim of the present study was to characterize the biomarker expression of these human colorectal cancer cell lines and analyze their resistance to oxaliplatin and irinotecan when exposed to low concentrations over short periods of time.2.Three human colorectal tumor cell lines were employed: SW-480 (CCL-228), SW-620 (CCL-227) and DLD-1 (CCL-221). All of them were purchased from American Type Culture Collection . The cells were cultured in Dulbecco´s modified Eagle´s medium  with 10% fetal bovine serum , 2 mM L-glutamine  and 1% penicillin/streptomycin  at 37 C in a humidified incubator  with 5% COThe expression of the different tumor biomarkers was analyzed by flow cytometry. A minimum of three replicates per biomarker were performed. The cells were grown in p100  for several days until exponential growth. Then, cells were digested with trypsin and washed twice with PBS 1X . The pellet was resuspended in PBS + BSA 0.1%  and incubated with the corresponding antibody for 15 min at room temperature in darkness. Next, the antibody was removed by centrifugation, and cells were finally detected in a FACS Canto cytometer .Anti-BCRP1, anti-CD133, anti-AC133, anti-EPCAM, anti-CD34, anti-CD36, anti-CD44V6 and anti-TROP2 antibodies were purchased from Miltenyi Company  and anti-LGR5 and anti-CD166 antibodies were purchased from Becton Dickinson . The dilution for each antibody is as described in the following 2O until 12 µL was incubated at 70 °C for 5 min. Then, 4 µL of buffer 5X, 1µL ribonuclease inhibitor, 2 µL 10 mM dNTP and 1 µL reverse transcriptase were added to each tube. The reaction mixture was then incubated at 25 °C for 5 min, 42 °C for 1 h and inactivated at 70 °C for 10 min. All cDNAs were stored at −20 °C until further use.Cells were harvested with trypsin and centrifuged. Total RNA from cultured cells was extracted using the RNeasy Mini kit  and used immediately for reverse transcriptase reactions or stored at −80 °C until use. RNA concentration was measured in a Nanodrop spectrophotometer . cDNA synthesis was performed using the RevertAid First Strand cDNA Synthesis kit . The reaction was prepared according to the manufacturer’s instructions: a mixture containing 1 µg of RNA, 1 µL of random hexamers and DEPC-HCD133, CD166, EpCAM and TACSTD2 using the KiCqStart SYBR Green kit  according to the manufacturer’s instructions. KiCqStart SYBR Green predesigned primers  were employed. All primers were used in a final concentration of 300 nM, and 10 ng of cDNA were used per well, for a total volume of 10 µL. All cDNA samples were measured in triplicate in a 96-well plate covered with adhesive seals in the thermocycler Roche LightCycler 480 . Reactions started with 10 min at 95 °C, followed by 45 cycles of 15 s at 95 °C, 1 min at 60 °C and 10 s at 72 °C. The 2−ΔCT method was used for calculating the normalized mRNA expression. Beta-actin was used as a housekeeping gene to normalize samples.The quantitative PCR reaction was performed for The cells were grown in p100 plates for several days until exponential growth. Then, two different plates were seeded: a control plate containing non-treated cells ) and a plate with treated cells . In the case of the DLD-1 cell line, a third plate was seeded with DMEM + 5 µg/mL oxaliplatin. The treated cells were exposed to this high dose of oxaliplatin (0.5 µg/mL or 5 µg/mL in the third DLD-1 plate) for 2 weeks and then maintained at half concentration . These treated and non-treated cells were then evaluated in the growth curve and viability assays.To analyze the growth, the cells were counted in a Neubauer chamber  at each passage. The total concentration and accumulated number of cells were calculated and represented in a graph.A thiazolyl blue tetrazolium bromide  assay was performed to detect the viability of the previously oxaliplatin-treated and non-treated cells exposed to different concentrations of oxaliplatin  and irinotecan  . Briefly, cells were seeded in 96-well plates  at a concentration of 200,000 cells/mL in a volume of 100 µL. Next, the chosen chemotherapeutic drug was added to each well. Three replicates per condition were performed in each plate with three independent experiments in total. After 72 h of the treatment, the medium was removed, and 50 µL of 0.5 mg/mL MTT was added. Then, plates were incubated for 4 h at 37 °C, and finally, the medium was removed and resuspended in 100 µL of dimethyl sulfoxide  before detection of absorbance in a BIOBASE-EL 10A  spectrophotometer at 546 nm.p-value < 0.05 was employed in all of the tests. GraphPad Prism 8  and FlowJo  were employed to perform the statistics and process the images. Finally, in order to determine the mathematical formulas connecting the decrease in viability with oxaliplatin concentration, we tested linear and exponential decay fits. The fits were performed in Python  with the statistical module “scipy.stats”. Since an exponential function looks like a straight line on a semilogarithmic plot, we performed a linear regression between the logarithm of the viability and the oxaliplatin concentration. A non-paired t test was used to compare two groups. One-way analysis of variance (ANOVA) was used to determine the differences between three groups. Wilcoxon test was used to compare the statistical differences between two paired groups, and the Friedman test to compare three or more paired groups. A The expression of biomarkers that have previously been related to tumorigenesis was quantified by flow cytometry in three human colorectal cell lines . Ten different biomarkers with a wide range of roles in cancer progression were analyzed. The mean of the percentages obtained and the standard error of each one is represented in A representation of the histograms obtained for each biomarker and each cell line is shown in PROM1), the SW-480 and SW-620 cell lines presented a higher significant expression when compared to DLD-1. This result corresponds with that obtained in the flow cytometry assay. In the case of CD166 (ALCAM), the SW-480 cell line expressed this biomarker the least, showing higher values in the SW-620 and DLD-1 cell lines. A similar profile was obtained by flow cytometry. EPCAM expression was also quantified, obtaining high values  in every cell line. However, the expression of EPCAM in DLD-1 was significantly higher than in the other cell lines. Finally, the TACSTD2 expression profile was similar tothe cytometry assay, with no expression in the SW-480 and SW-620 cell lines and significantly different expression in DLD-1.Finally, the expression of biomarkers that showed differences between cell lines in the previous flow cytometry experiment was confirmed via RT-qPCR assays. Normalized expression is shown in The growth of colorectal cell lines differed when they were exposed to the oxaliplatin chemotherapy drug. Cells were seeded for 2 weeks in exposure to oxaliplatin; the number of cells for each passage is represented in The resistance to oxaliplatin was measured in the SW-480, SW-620 and DLD-1 cell lines, using previously oxaliplatin-treated and non-treated cells. The resistance to oxaliplatin was evaluated in a wide range of oxaliplatin concentrations (0–50 µg/mL).Regarding the SW-480 and SW-620 non-treated cell lines , gradualThe DLD-1 non-treated cell line  showed lp-values , meaning the cell viability decreased exponentially at low and high ranges of oxaliplatin concentrations.In order to determine the mathematical formula relating the decrease in viability to the oxaliplatin concentration, we tested linear and exponential decay fits. Two ranges of oxaliplatin concentrations were evaluated: 0–0.4 µg/mL and 3.25–25 µg/mL in SW-480 and SW-620, and 0–0.4 µg/mL and 12.5–50 µg/mL in DLD-1. These concentrations were used because cell viability was maintained in the intermediate range, as previously described. The exponential fits  reportedRegarding the previously oxaliplatin-treated cells, a profile similar to that of the control non-treated cells was obtained. Despite this similar profile, there were significant differences between non-treated and previously oxaliplatin-treated cells in all cell lines. The cell viability percentages were significantly higher in previously oxaliplatin-treated cells when compared to control non-treated cells a, which All of these results led us to conclude that previously oxaliplatin-treated cells were resistant to the same drug to which they were exposed  but not to others (irinotecan).TACSTD2 increased or not in previously exposed cells to evaluate the role of TROP2 in the resistance to oxaliplatin. TACSTD2 versus beta-actin (reference gene). The SW-480 and SW-620 cell lines did not present TACSTD2 expression in non-treated or treated cells. However, significant differences were found between previously oxaliplatin-treated  and non-treated DLD1 cells.We have already shown that TROP2 is the biomarker with high expression in DLD1, with this line being the most resistant to oxaliplatin. Moreover, we have demonstrated that cells previously exposed to oxaliplatin are more resistant than non-exposed cells. In this section, we wanted to test whether the expression of The present work aimed to characterize tumor biomarker expression (diagnostic tool) and the response to chemotherapy (resistance to drugs) of human colorectal cell lines. We selected three different colorectal cell lines: the first from a primary tumor, the second from a metastatic tumor, and the third characterized by its resistance to chemotherapeutic drugs. We showed the differential distribution of a variety of controversial biomarkers’ expression in three representative cell lines with different tumor profiles. Ten different biomarkers related to metastasis, invasion, and adhesion were chosen. All of them had been previously described in the literature as cancer stem cell biomarkers, although their specific roles are still under debate . AlthougIn our experiments we showed that the SW-480 and SW-620 cell lines presented a similar expression profile, but CD166 was overexpressed in SW-620. SW-480 proceeded from a primary adenocarcinoma cancer, while SW-620 came from the metastasis in the same patient. This indicates that CD166 may play an important role in metastasis. This was confirmed by studies that support that this biomarker is capable of promoting metastasis by interacting with other molecules, such as SOSTDC1 ,38. MoreTACSTD2) in the DLD-1 cell line when compared to that in the SW-480 and SW-620 cell lines, which were more resistant to chemotherapy. Moreover, we demonstrated that previously oxaliplatin-exposed cell lines are more resistant to oxaliplatin and, in the case of DLD-1, present higher expression of TACSTD2. Although further experiments are needed, all of these results together show a correlation between the expression of this protein and a drug-resistant phenotype, a promising result for predicting the response to oxaliplatin in patients.Another significant difference was found in relation to the Trop2 protein. Trop2 is a transmembrane glycoprotein that transduces its signal by means of calcium upon binding to PIP2 in its intracellular domain. In this study, we evaluated its expression and its relation to drug resistance. We showed a significantly higher expression of TROP2  pathway and irinotecan resistance has been related to epigenetic modifications such as histone acetylation . This isTo conclude, we found differences in the expression of biomarkers that allowed us to discern between different tumorigenicity profiles. On the other hand, exposure to oxaliplatin slows cell growth but generates resistance to it. The tumor microenvironment, tumor malignancy and the EMT capacity can condition both protein expression and cell growth in response to resistance. Further analyses in this area will provide important information for improved understanding of the cellular response of different colorectal tumors to chemotherapy, their resistance to treatment and how to prevent it.


Isobolographic analysis allowed for the assessment of the interactions between the studied compounds. Importantly, fraxetin was not cytotoxic to normal keratinocytes (HaCaT) and melanocytes (HEMa-LP), although it slightly inhibited their viability at high concentrations. The combination of fraxetin with cisplatin and mitoxantrone showed the additive interaction, which seems to be a promising direction in melanoma therapy. Unfortunately, the combination of fraxetin with docetaxel may not be beneficial due to the antagonistic antiproliferative effect of both drugs used in the mixture.Malignant melanoma is a skin cancer characterized by rapid development, poor prognosis and high mortality. Due to the frequent drug resistance and/or early metastases in melanoma, new therapeutic methods are urgently needed. The study aimed at assessing the cytotoxic and antiproliferative effects of scoparone and fraxetin in vitro, when used alone and in combination with three cytostatics: cisplatin, mitoxantrone, and docetaxel in four human melanoma cell lines. Our experiments showed that scoparone in the concentration range tested up to 200 µM had no significant effect on the viability of human malignant melanoma , while fraxetin inhibited cell proliferation with IC Malignant melanomas are neoplasms derived from melanocytes and are characterized by early metastasis, rapid development, poor prognosis, and high mortality . The morAmong the numerous chemotherapeutic agents used, we can distinguish mitoxantrone (MTX), docetaxel (DOCX), or cisplatin (CDDP). Mitoxantrone is a synthetic anthraquinone derivative with an anti-cancer effect that inhibits DNA synthesis and transcription. MTX binds to nucleic acids via intercalation and induces apoptosis in both protein-bound DNA and DNA unrelated to the DNA chain breakage site . MTX hasDocetaxel (DOCX) is one of the taxanes, a diterpene alkaloid, and it belongs to tubule stabilizing drugs . The staCisplatin (CDDP) is one of the most powerful and widely used anti-cancer drugs that is clinically effective in the treatment of a variety of solid tumors, including ovarian cancer, adrenal cortex cancer, and malignant melanoma . CisplatSuch compounds may be, e.g., coumarins, which are a large class of natural phenolic compounds of plant origin. Coumarins have a variety of pharmacological effects, including antithrombotic, anti-inflammatory, antibacterial, and especially antitumor, antioxidant, and neuroprotective effects ,19,20. IArtemisia scoparia and has been shown to relax smooth muscles, reduce total cholesterol and triglycerides, and delay specific pathomorphological changes in rabbits with hypercholesterolaemia and diabetes. Various properties of scoparone, including the ability to scavenge reactive oxygen species, inhibition of tyrosine kinases, and enhancement of prostaglandin production, have been suggested to account for these findings [One of the simple coumarins is scoparone , which has been purified from the Chinese herb findings ,24,25. Afindings ,27, and findings ,29. In aFraxinus bungeana and Fraxinus rhynchophylla. It shows many biological activities, including antibacterial, anti-inflammatory, neuroprotective [Fraxetin  is a coumarin originally isolated from the plants otective ,31,32,33otective ,35. FraxFRX, SCP, CDDP, MTX, and DOCX inhibited the viability of human melanoma cell lines, both primary  and metastatic , in a concentration-dependent manner when applied separately. Incubation of FM55P, FM55M2, A375, and SK-MEL 28 cells with increasing concentrations of FRX resulted in a reduction in melanoma cell viability . ScoparoCytotoxicity of fraxetin (FRX) to normal human keratinocytes (HaCaT), normal human melanocytes (HEMa-LP), and malignant melanoma cells  was measured by LDH assay. The LDH test allowed for the assessment of the release of lactate dehydrogenase into the medium, which indicates damage to the cell membrane and cell death . In our Fraxetin (FRX) administered alone dose-dependently reduced the proliferation of FM55P, FM55M2, A375, and SK-MEL 28 melanoma cells. The treatment of all the investigated cancer cells with increasing concentrations of FRX reduced DNA synthesis which was evaluated by measuring BrdU (5-bromo-2′-deoxyuridine) incorporation into cellular DNA in proliferating cells . As pres50 values ± SEM). The experimentally derived median inhibitory concentration (IC50) values for FRX were: 32.42 ± 4.21 µM for FM55P, 46.04 ± 4.17 µM for FM55M2, 44.03 ± 12.02 µM for A375, and 73.16 ± 7.38 µM for SK-MEL 28, respectively. The IC50 values for scoparone (SCP) and fraxetin (FRX) are shown in Next, we analyzed the concentration-dependent inhibitory effects of CDDP, MTX, and DOCX on melanoma cells to find out if their combinations with FRX could enhance the antitumor effects of these chemotherapeutic agents. The equations of log-probit concentration–response relationship curves (CRRC) for the studied drugs when used alone and for the combinations of CDDP with FRX, MTX with FRX, and DOCX with FRX  allowed 50) ranging from 1.3 to 3.3 µM for CDDP, from 0.03 to 1.7 µM for MTX, and from 1.27 to 15.87 nM for DOCX were mentioned in previous experiments [The experimentally derived mean inhibitory concentration  are mutually non-parallel to each other in FM55P, FM55M2, A375, and SK-MEL28 cell lines. The exception is the combination of FRX with MTX for the A375 line C’, whereIsobolographic analysis of the interaction between FRX and CDDP at the fixed ratio of 1:1 showed additivity in the studied melanoma cell lines   and addiThe combination of FRX and MTX at the fixed ratio of 1:1 showed additive interactions for FM55P, A375, and SK-MEL28 , Table 3In contrast, the combination of FRX with DOCX at the fixed ratio of 1:1 produced antagonistic interaction in the A375 cell line and additivity with a tendency to antagonistic interaction in other melanoma cell lines tested .Malignant melanoma is a global problem due to the steady increase in the incidence of the disease worldwide. In 2020, nearly 325,000 cases of melanoma were diagnosed in patients globally and nearly 58,000 people died . It is f50 = 41.3 µM/L, although in the case of the other PC-3 prostate cancer cell line its inhibitory effect was small. Nevertheless, both cell lines showed reduced proliferation at higher scoparone concentrations (>100 µM/L). In addition, researchers found that high doses of scoparone inhibited the proliferation of breast cancer cell lines (MCF-7 and MDA-MB-231), cervical cancer (HeLa), hepatocellular carcinoma (HepG2 and Hep3B), and colon cancer . In the DU145 prostate cancer line, against which scoparone was the most potent, it was due to cell cycle arrest in the G1 phase without inducing apoptosis. Scoparone inhibited the transcriptional activity of STAT3 and thus suppressed the transcription of oncogenic STAT3 target genes, leading to inhibition of the growth of prostate cancer cells [Scoparone, one of the ingredients of herbal Chinese medicine, has a number of pharmacological effects; therefore, it is used in the treatment of many diseases. Its action depends on the absorption and metabolism of this compound. It has been shown that, in human liver microsomes, scoparone is oxidized to isoscopoletin via 6-O-demethylation. The oxidation rate is about 0.2–0.4 µmol/(minx g of protein) and is related to the functioning of CYP2A13, which shows the highest rate of oxidation. Scoparone and its derivatives are completely excreted in the urine within 24 h . Scoparoer cells .In the case of studies on scoparone in relation to melanoma, it was shown that this compound at a concentration of 50 µg/mL caused almost an 8.5-fold increase in cell melanin content compared with control cells but had no effect on the proliferation of murine B16 melanoma cells . Our exp50 doses obtained in this study in the range of 32.42–73.16 µM are comparable. Zhand et al. showed that fraxetin also inhibits the growth of non-small cell lung cancer cells with IC50 doses of 20.12 µM and 22.45 µM for HCC827 and H1650 lines, respectively. Even at a high concentration of fraxetin (100 µM), it did not show any inhibition of the growth of normal lung cells. Fraxetin inhibited IL-6-induced phosphorylation of tyrosine residue (Tyr705) signal transducer and activator of transcription 3 (STAT3). FRX interacts with STAT3 through hydrogen bounding and hydrophobic interaction [Fraxetin seems to be a much more promising natural compound in anti-cancer therapy. It was shown that fraxetin inhibited the proliferation of colon adenocarcinoma cells (cell lines: HCT116 and DLD-1). Its action was related to the arrest of the cell cycle in the S phase and the induction of cell apoptosis. Decreased levels of p-JAK2 after fraxetin application were observed via the JAK2/STAT3 signaling pathway. Additionally, fraxetin was characterized by low toxicity . Numeroueraction . The anteraction . In addieraction . Fraxetieraction .Fraxetin, apart from its anti-cancer properties, has anti-inflammatory, hepatoprotective, and anti-fibrotic properties. An interesting feature of fraxetin is its antioxidant effect ,48. Expe50 dose was about 67.25–89.58 µM depending on the cell line types. The experiments also confirmed that the combination of the most preferred osthole with cisplatin gave an additive and synergistic interaction depending on the melanoma cell line tested. The combination of CDDP with xanthotoxol and imperatorin caused an additive interaction, while the combination of CDDP with xanthotoxin and isopimpinellin, which was not very effective against melanoma, resulted in an antagonistic interaction [Generally, coumarins are characterized by numerous biological effects, some of which are used in medicine. There are many reports of their anti-cancer activity, but each tumor responds to specific coumarins differently . For exaeraction . Our experaction . Our preTwo cell lines of primary melanoma, A375 and FM55P, and two lines of metastatic melanoma, SK-MEL28 and FM55M2, were used in the experiments. A375 and SK-MEL28 cell lines were from the American Type Culture Collection , and FM55P and FM55M2 lines were from the European Collection of Cell Cultures . The growth conditions of the culture were described earlier ,38.2+ and Mg2+. Mitoxantrone (MTX—Sigma-Aldrich) and docetaxel (DOCX—Sigma-Aldrich) were dissolved in DMSO as stock solutions. The examined coumarins, fraxetin (FRX) and scoparone (SCP)—, were dissolved in DMSO as stock solutions. The drugs were dissolved to the respective concentration with culture medium before their use. PBS and DMSO had no effect on cell proliferation.Cisplatin  was dissolved in phosphate buffered saline (PBS) with Ca4 cells/mL, 2 × 104 cells/mL, 2 × 104 cells/mL, and 3 × 104 cells/mL, respectively. The next day, the culture medium was removed and cells were exposed to serial dilutions of FRX, SCP, CDDP, MTX, and DOCX, in fresh culture medium. The next steps of the MTT test have been described by us earlier [Cell viability was determined using the MTT assay. FM55P, FM55M2, A375, and SK-MEL 28 cells were placed on 96-well plates  at a density of 2 × 10 earlier . Each trThe cell proliferation BrdU test was performed by the ELISA method using a kit . Optimized amounts of cells (as described above) were placed on a 96-well plate (Nunc) (100 µL/well). On the next day, the cancer cells were treated with increased concentrations of FRX for 48 h, followed by 10 µL/well BrdU Labeling Solution (100 µM), and then cells were reincubated for an additional 24 h at 37 °C. Then, the BrdU assay was performed following the manufacturer’s instructions. Quantitation was performed spectrophotometrically at 450 nm using a microplate spectrophotometer .Optimized amounts of malignant melanoma cells (as above), normal human keratinocytes HaCaT (1 × 104/mL), and normal human melanocytes HEMa-LP (5 × 103/mL) cells were placed on 96-well plates (Nunc). The next day, the cells were washed in PBS and then exposed to increasing concentrations of FRX in fresh culture medium. The cytotoxicity was estimated based on the measurement of cytoplasmic lactate dehydrogenase (LDH) activity released from damaged cells after 72 h of exposure to FRX. The LDH assay was performed according to the manufacturer’s instruction (Cytotoxicity Detection KitPLUS LDH) (Roche). More information about the steps of the LDH assay has been described earlier ,38.50 values) of FRX and CDDP, MTX, and DOCX were calculated. The test for parallelism between two concentration–response curves  was performed as described in our previous studies [50 values for the drugs administered alone, median additive inhibitory concentrations for the mixture of FRX + CDDP, FRX + MTX, and FRX + DOCX at the fixed ratio of 1:1 (IC50add) were calculated, as described earlier [50 exp values for the mixture of FRX with CDDP, FRX with MTX, and FRX with DOCX (at the fixed ratio of 1:1) were determined based on the concentrations of the mixtures of CDDP or MTX or DOCX with FRX, inhibiting 50% of cell viability in the melanoma cell lines  measured in vitro by the MTT assay. Details concerning the isobolographic analysis have been published elsewhere [Isobolographic analysis is a statistical method allowing the determination of precise characteristics of pharmacodynamic interactions between drugs and chemical substances. This method is the gold standard among drug interaction assessment methods. Log-probit analysis according to Litchfield and Wilcoxon  was used studies ,54,55,56 earlier ,58. The lsewhere ,60.p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). The IC50 values for FRX, CDDP, MTX, and DOCX, when administered alone, and the IC50exp values for the combinations at the fixed ratio of 1:1 were calculated by computer-assisted log-probit analysis, as described elsewhere [50exp values for the mixture of FRX with CDDP, FRX with MTX, and FRX with DOCX were statistically compared with their respective theoretically additive IC50add values by the use of an unpaired Student’s t-test, as reported earlier [GraphPad Prism 8.0 Statistic Software was used for statistical analysis of data. The calculations were performed by one-way analysis of variance (ANOVA test) for multiple comparisons followed by Tukey’s significance test. Data are expressed as the mean ± standard error (SEM)  and mitoxantrone (MTX) shows an additive interaction, which seems to be a promising approach in melanoma therapy. Unfortunately, the combination of fraxetin with docetaxel may not be beneficial due to the antagonistic antiproliferative effect of both drugs used in the mixture. Further intensive experiments are needed to determine the possible mechanisms of action of the combinations of coumarins with cytostatics and to conduct animal experiments to confirm the in vivo interactions of the compounds.


To achieve personalized 64Cu-ipRIT, we developed a new in vitro tumor cell-binding assay (64Cu-TuBA) system with a panel containing nine candidate 64Cu-labeled antibodies targeting seven antigens , which are reportedly overexpressed in patients with pancreatic cancer. We investigated the feasibility of 64Cu-TuBA to select the highest-binding antibody for individual cancer cell lines and predict the treatment response in vivo for 64Cu-ipRIT. 64Cu-TuBA was performed using six human pancreatic cancer cell lines. For three cell lines, an in vivo treatment study was performed with 64Cu-ipRIT using high-, middle-, or low-binding antibodies in each peritoneal dissemination mouse model. The high-binding antibodies significantly prolonged survival in each mouse model, while low-and middle-binding antibodies were ineffective. There was a correlation between in vitro cell binding and in vivo therapeutic efficacy. Our findings suggest that 64Cu-TuBA can be used for patient selection to enable personalized 64Cu-ipRIT. Tumor cells isolated from surgically resected tumor tissues would be suitable for analysis with the 64Cu-TuBA system in future clinical studies.Peritoneal dissemination of pancreatic cancer has a poor prognosis. We have reported that intraperitoneal radioimmunotherapy using a  Pancreatic cancer has a dismal prognosis, with an overall 5-year survival rate below 10% ,3,4. Sur64Cu-labeled antibody (64Cu-ipRIT). Generally, RIT has several advantages over immunotherapy; e.g., RIT can target and kill cancer cells by irradiation from radionuclides bound to antibodies and does not require a functional immune system [64Cu-labeled anti-epidermal growth factor receptor (EGFR) antibody cetuximab (64Cu-cetuximab) to investigate the efficacy of 64Cu-ipRIT. Cetuximab has a high binding affinity for EGFR, which is reportedly overexpressed in >90% of pancreatic cancers [64Cu shows β– decay , electron capture (42.6%), and β+ decay ; thus, 64Cu can be used for internal radiotherapy, as well as for positron emission tomography (PET) imaging. For therapeutic use, β– particles and Auger electrons emitted from 64Cu damage tumor cells, with the high-linear energy transfer Auger electrons causing heavy damage to cancer cell DNA [64Cu-cetuximab was shown to be effective in inhibiting local recurrence and regrowth of distant metastasis, including peritoneal dissemination and liver metastasis. It also significantly prolonged survival with little toxicity, as observed using an orthotopic xenograft mouse model after surgery to resect primary pancreatic tumors [64Cu-ipRIT using another high-binding 64Cu-labeled antibody. To accomplish this personalized approach for 64Cu-ipRIT, the selection of a high-binding antibody for individual cancers prior to treatment is important.To address this, we focused on intraperitoneal radioimmunotherapy using a e system . We deve cancers . 64Cu shcell DNA . The ipRc tumors ,12. HoweWestern blotting has been widely used as a method to investigate the relative protein expression levels of a certain target antigen and compare these among individual tumors ,14. Howe64Cu-ipRIT. However, the correlation between in vitro antibody binding and in vivo therapeutic efficacy for 64Cu-ipRIT remains unclear.Recently, in vitro assays with tumor cells or primary tumor cell cultures, obtained from individual resected tumor tissues, have been actively studied, and their usefulness as a tool for selecting optimal drugs or antibodies in personalized chemotherapy or molecular-targeted therapy has also been evaluated . Therefo64Cu-TuBA system, using a panel containing nine candidate 64Cu-labeled antibodies: 64Cu-anti-EGFR antibodies (cetuximab and panitumumab), anti-HER2 antibodies (trastuzumab and pertuzumab), anti-HER3, anti-TfR, anti-EpCAM, anti-LAT1, and anti-CD98 antibodies , which are reportedly overexpressed in pancreatic cancer patients [64Cu-labeled antibodies to perform the in vitro tumor cell-binding assay as those used in 64Cu-ipRIT because this would result in a better prediction of in vivo efficacy by in vitro assay.Here, we investigated the feasibility of using an in vitro tumor cell-binding assay to select the optimal antibody for an individual cancer and to predict treatment response in vivo, using human pancreatic cancer cell lines. As a proof-of-concept, we developed a new in vitro tumor cell-binding assay system, the tibodies . To estapatients ,22,23,2464Cu-labeled antibodies, including 64Cu-anti-EGFR antibodies (cetuximab and panitumumab), anti-HER2 antibodies (trastuzumab and pertuzumab), anti-HER3 antibodies, anti-TfR antibodies, anti-EpCAM antibodies, anti-LAT1 antibodies, and anti-CD98 antibodies. We then conducted an in vitro cell-binding assay with this 64Cu-labeled antibody panel with six human pancreatic cancer cell lines  , whereas Capan-1 showed low cell binding (9.2%). There were significant differences between these cell lines  (p < 0.05). 64Cu-cetuximab showed higher cell binding than 64Cu-panitumumab in all cell lines (p < 0.05). Capan-1 showed higher cell binding for 64Cu-anti-TfR antibody (23.2%) than 64Cu-cetuximab (9.2%) (p < 0.05). The other 64Cu-labeled antibodies, 64Cu-anti-HER2 antibodies (trastuzumab and pertuzumab), anti-HER3, anti-TfR, anti-EpCAM, anti-LAT1, and anti-CD98 antibodies showed low cell binding in all cell lines. In addition, we observed a correlation between EGFR expression as measured by Western blotting and cell binding (%) , but the coefficient of variation (CV) of Western blotting was significantly greater than that of cell binding (%) (p < 0.05). Thus, the in vitro cell-binding assay showed smaller variations than Western blotting.The schematic of this study is shown in d PSN-1) . The 64Cding (%)  , 64Cu-anti-TfR antibody, and 64Cu-anti-CD98 antibody, and was determined to be high, medium, or low binding for each cell line. The survival curves are shown in 64Cu-ipRIT with 64Cu-anti-EGFR antibody (cetuximab), showing high binding for AsPC-1 cells, was greater than that observed for the saline control (p = 0.0025). On the other hand, there were no significant differences in survival after 64Cu-ipRIT with 64Cu-anti-TfR antibody and 64Cu-anti-CD98 antibody, showing moderate and low binding for AsPC-1 cells compared to the saline control. For the peritoneal dissemination mouse models of PSN-1, survival after 64Cu-ipRIT with 64Cu-anti-EGFR antibody (cetuximab) and 64Cu-anti-TfR antibody, showing high and middle binding for PSN-1 cells, was greater than that observed for the saline control . However, no significant difference was found for the 64Cu-anti-CD98 antibody, showing low binding for PSN-1 cells compared to the saline control. The peritoneal dissemination mouse models of Capan-1 with 64Cu-anti-TfR antibody showed high binding for Capan-1 cells, which was higher than that of the saline control (p = 0.0142). Comparatively, no significant difference was detected in 64Cu-cetuximab and 64Cu-anti-CD98 antibodies, showing middle-low binding for Capan-1 cells compared with the saline control. The mean survival time (MST) and %MST are summarized in 64Cu-cetuximab > 64Cu-anti-TfR antibody > 64Cu-anti-CD98 antibody > saline control in AsPC-1 and PSN-1, while the values were 64Cu-anti-TfR antibody > 64Cu-cetuximab > 64Cu-anti-CD98 antibody > saline control in Capan-1. In the in vivo treatment study, all mice in each group reached a humane endpoint due to noticeable extension of the abdomen due to tumor growth in the peritoneum. After treatment, there was no weight loss of more than 20% compared with the initial body weight due to drug administration in any treatment group for all cell line models .For the in vivo study, peritoneal dissemination mouse models with three pancreatic cancer cell lines, AsPC-1, Capan-1, and PSN-1, were used. The efficacy of e models . Figure 64Cu-TuBA systems selected high-binding antibodies for individual cancer cell lines and predicted treatment response in each peritoneal dissemination mouse model. These findings indicated the feasibility of 64Cu-TuBA and suggested that this method would be useful to enable personalized 64Cu-ipRIT. Pancreatic cancer has one of the poorest prognoses among all types of cancer [64Cu-cetuximab was effective as adjuvant therapy after surgery for pancreatic cancer in vivo [64Cu-ipRIT for use as a postoperative adjuvant therapy, an in vitro tumor cell-binding assay can be conducted with tumor cells obtained from isolated pancreatic cancer samples immediately after surgery. Then, based on the in vitro tumor cell-binding assay with individual patient tumor cells, a high-binding 64Cu-labeled antibody can be selected and used for the 64Cu-ipRIT adjuvant therapy. Since 64Cu-TuBA was able to select high-binding 64Cu-labeled antibodies in all the examined pancreatic cancer cell lines, this method should provide optimal antibodies to most pancreatic cancer patients.We demonstrated that f cancer ,2,3,4,5. in vivo . Assumin64Cu-labeled antibodies targeting seven antigens as a proof-of-concept study of 64Cu-TuBA. Of the examined antibodies, those for EGFR and TfR showed relatively high binding to the pancreatic cancer cells used in this study, compared with those for HER2, HER3, EpCAM, LAT1, and CD98. Our results suggest that this assay can be easily applied to a variety of antibodies. For the future clinical use of this assay, it would be beneficial to add the other candidate antibodies used in the panel. Antibody arrays, which have been variously developed for biomarker detection in pancreatic cancer [64Cu-TuBA format in the future. Thus far, no pancreatic cancer-specific therapeutic antibodies have been approved for the treatment of pancreatic cancer despite the effort of previous studies; that is, it is difficult to cover most pancreatic cancer patients by targeting one specific target antibody [64Cu-ipRIT with 64Cu-TuBA could be a beneficial strategy for pancreatic cancer treatment. We used 64Cu-labeled antibodies, rather than antibodies with other types of labels, for the in vitro tumor cell-binding assays as a proof-of-concept study, since these are the same compounds used in 64Cu-ipRIT. This study successfully demonstrated the correlation between binding ability in in vitro cell-binding assays with 64Cu-labeled antibodies and in vivo therapeutic efficacy of 64Cu-ipRIT. Thus, this is an important basic finding to advance further development of personalized 64Cu-ipRIT. To make this assay easier to use in various locations, replacing 64Cu-labeled antibodies with fluorescent-labeled antibodies might be worth exploring in future studies. However, it is necessary to note the differences between 64Cu-labeling and fluorescent-labeling, such as binding affinity and labeling efficiency.We used a panel containing nine candidate c cancer ,37, mighantibody . Therefo64Cu-ipRIT with 64Cu-cetuximab was effective in AsPC-1 and PSN-1, while 64Cu-anti-TfR antibody was effective for Capan-1 in each peritoneal dissemination mouse model. In these cases, cell binding (%) showed 44.7 ± 3.0% and 31.2 ± 0.3% for 64Cu-cetuximab in AsPC-1 and PSN-1 and 23.2 ± 1.1% for 64Cu-anti-TfR antibody in Capan-1. Thus, it could be effective in vivo, at least when cell binding (%) is more than 20%, as in this experiment. In this in vivo experiment, we observed significant increases in survival time with respective treatment with high-binding antibodies for each cell line model. However, all models subsequently recurred. In our previous study, we showed that vorinostat, a histone deacetylase inhibitor, is an effective radiosensitizer for use in the treatment of peritoneal dissemination of gastric cancer by ipRIT with 64Cu-cetuximab [64Cu-ipRIT for the treatment of peritoneal dissemination of pancreatic cancer.From the in vivo treatment study, we found that there was a strong correlation between cell binding (%) from the in vitro study and the relative survival time in all examined cell line models. These findings suggest the feasibility of an in vitro tumor cell-binding assay to observe not only cell binding, but also predict the therapeutic efficacy in vivo. We observed that etuximab . This su64Cu-ipRIT (22.2 MBq/mouse), which was determined by previous studies with 64Cu-cetuximab, for comparison. This was optimized as the maximum tolerated dose of 64Cu-cetuximab, which was the best antibody for in vivo treatment in the present study. The optimal doses for each 64Cu-labeled antibody will be evaluated in future clinical trials. Second, in the present study, we used peritoneal dissemination mouse models for the in vivo studies. In clinical practice, most pancreatic cancer patients show other types of recurrence, such as local recurrence and hepatic metastasis, as well as peritoneal dissemination after surgery [64Cu-ipRIT with 64Cu-cetuximab reduces local recurrence and hepatic metastasis [64Cu-labeled antibodies against other types of recurrence in future preclinical and clinical studies.This study has several limitations. First, the present study used one fixed administration dose for the in vivo treatment of  surgery ,5,6. Alttastasis , it is n64Cu was produced on a cyclotron at the National Institutes for Quantum and Radiological Science and Technology  and purified using previously published methods [64Cu-PCTA-antibodies were prepared using methods reported in a previous study [64Cu-labeled antibody panel was prepared before the in vitro cell-binding assay, as shown in Antibodies used in this study and the sources are listed in  methods . Accordi methods , the bifus study , with a 2 at 37 °C. RPMI 1640 (Wako) supplemented with 10% fetal bovine serum (FBS) was used for AsPC-1, BxPC-3, and PSN-1. On the other hand, DMEM  supplemented with 10% FBS was used for MIA PaCa-2 and PANC-1, while IMDM  supplemented with 20% FBS was used for Capan-1. Exponentially growing cells were detached from the culture plates with trypsin and used in this study. The number of viable cells was determined using the trypan blue dye exclusion method.Human pancreatic cancer cell lines  were obtained from the American Type Culture Collection . The cells were cultured in a humidified atmosphere of 5% CO64Cu-labeled antibody panel. For the cell-binding assay, 3 × 105 cultured cells from each cell line were diluted in 1 mL of ice-cold phosphate-buffered saline (PBS) with 1% bovine serum albumin (BSA) (Sigma-Aldrich) and were added to 2 mL centrifuge tubes and incubated with each 64Cu-labeled antibody (20 kBq) on ice for 1 h. Then cells were washed with ice-cold PBS on ice. After washing, the radioactivity bound to the cells was measured using a γ-counter . The percentage of cell binding was calculated as (radioactivity of the collected cells/radioactivity administered to the cells × 100) (%).An in vitro cell-binding assay was performed using the 5 cells, n = 3 for each cell line) were lysed with lysis buffer containing protease inhibitor cocktail  according to the manufacturer’s protocol, and protein concentrations were determined using a BCA protein assay kit . The SDS-polyacrylamide gel electrophoresis was performed with 15 μg of protein in each sample using 5–20% gel (ATOO) and transferred to a PVDF membrane . After blocking in 0.05% Triton-TBS containing 1% BSA  at room temperature for 30 min, the membrane was incubated at 4 °C overnight with each primary antibody. For the primary antibodies, rabbit anti-EGFR antibodies  and mouse anti-GAPDH antibodies  were used as loading controls. Then, the excess antibody was washed with 0.05% TBST, and the membrane was incubated with the secondary antibody ) at room temperature for 2 h. After washing, the membrane was incubated with SuperSignal West Pico Chemiluminescent Substrate  at room temperature for 5 min, and signals were detected using an X-ray film. After exposure, the membrane was incubated in stripping buffer at 37 °C for 30 min to strip off the former antibody. The intensity was calculated by densitometry using the ImageJ software .Protein expression levels of EGFR were examined by Western blot analysis and compared with values of the cell binding (%) obtained from the cell-binding assay in each cell line. The cultured cells (3 × 106) suspended in 500 µL phosphate buffered saline (PBS) were injected intraperitoneally 1 week before treatment which resulted in the small nodules of peritoneal dissemination 1 week after cell inoculation.Six-week-old female NOD.CB17-Prkdc SCID/J mice  were obtained from Charles River Laboratories  and were used in this study. Before the experiments, the mice were acclimated for at least 1 week. All animal experimental procedures were approved by the Animal Ethics Committee of the National Institutes for Quantum Science and Technology and conducted in accordance with the institutional guidelines. To generate peritoneal dissemination mouse models, AsPC-1, Capan-1, and PSN-1 cell lines were used. Cells , 64Cu-anti-TfR antibody, and 64Cu-anti-CD98 antibody using peritoneal dissemination mouse models of AsPC-1, Capan-1, and PSN-1 cell lines. Mice with peritoneal dissemination were randomized into four groups for each cell line (n = 7/group). Mice were injected intraperitoneally with 22.2 MBq 64Cu-anti-EGFR antibody (cetuximab), 64Cu-anti-TfR antibody, or 64Cu-anti-CD98 antibody  . For comparison, mice were examined after administration of saline  . The dose of 64Cu-ipRIT was determined based on a previous report [The efficacy of s report . The micp < 0.05.Data were expressed as means with corresponding standard deviations. Multiple comparisons were conducted using one-way analysis of variance (ANOVA) with post hoc comparisons using the Tukey–Kramer test. Differences in survival were evaluated using log-rank tests. Statistical significance was set at 64Cu-TuBA, to select antibodies with high binding affinity for individual cancer and to predict treatment response to 64Cu-ipRIT. This method would enable individual patients with pancreatic cancer to receive the optimal treatment, leading to better patient care and lower costs.This study demonstrated the feasibility of an in vitro tumor cell-binding assay,