Prostate cancer is a broad-spectrum disease, spanning from indolent to a highly aggressive lethal malignancy. Prostate cancer cell lines are essential tools to understanding the basic features of this malignancy, as well as in identifying novel therapeutic strategies. However, most cell lines routinely used in prostate cancer research are derived from metastatic disease and may not fully elucidate the molecular events underlying the early stages of cancer development and progression. Thus, there is a need for new cell lines derived from localised disease to better span the disease spectrum.Prostatic tissue from the primary site, and adjacent non-cancerous tissue was obtained from four patients with localised disease undergoing radical prostatectomy. Epithelial cell outgrowths were immortalised with human papillomavirus type 16 (HPV16) E6 and E7 to establish monoclonal cell lines. Chromosomal ploidy was imaged and STR profiles were determined. Cell morphology, colony formation and cell proliferation characteristics were assessed. Androgen receptor (AR) expression and AR-responsiveness to androgen treatment were analysed by immunofluorescence and RT-qPCR, respectively. RNA-seq analysis was performed to identify prostate lineage markers and expression of prostate cancer tumorigenesis-related genes.Two benign cell lines derived from non-cancer cells (AQ0420 and AQ0396) and two tumour tissue derived cancer cell lines (AQ0411 and AQ0415) were immortalised from four patients with localised prostatic adenocarcinoma. The cell lines presented an epithelial morphology and a slow to moderate proliferative rate. None of the cell lines formed anchorage independent colonies or displayed AR-responsiveness. Comparative RNA-seq expression analysis confirmed the prostatic lineage of the four cell lines, with a distinct gene expression profile from that of the metastatic prostate cancer cell lines, PC-3 and LNCaP.Comprehensive characterization of these cell lines may provide new in vitro tools that could bridge the current knowledge gap between benign, early-stage and metastatic disease. Prostate cancer was diagnosed in over 1.4 million men in 2020, and approximately 375,000 associated deaths were recorded worldwide in the same year [LNCaP, PC-3 and DU145 are the most commonly used in vitro cell lines for prostate cancer research –4 and arIn conjunction with industry collaborators, we developed four cell lines from prostates surgically removed as part of localised prostate cancer management to bridge this gap. Two epithelial prostate cell lines were derived from the primary tumour and two from adjacent benign tissue. In this study, we describe some of the main molecular and phenotypic characteristics of these immortalised cell lines.Prostate tissue derived from the primary tumour or adjacent benign tissue was obtained from men undergoing radical prostatectomy for the management of localised prostate cancer in 2007. Cell lines were developed in collaboration with the now ceased UK-based biotechnology company Onyvax Ltd. Briefly, fresh tissues were minced and seeded in proprietary Onyvax medium to support epithelial cell growth. Cell lines were developed from outgrowths of these prostate epithelial cells in Keratinocyte Serum-Free Growth Medium media , and transformed with replication-defective human papillomavirus type 16 (HPV16) E6 and E7. Briefly, at passage 3, the propagated primary cells were infected with the HPV16 E6/E7 virus in the presence of 8 µg/mL of polybrene, as reported previously . For subD5S818, D13S317, D7S820, D16S539, vWA, TH01, TPOX, CSF1PO and D21S11), and the amelogenin sex-determining marker (AMEL), were amplified using the Applied Biosystems 3130XL Genetic Analyser platform (ThermoFisher Scientific). The genotype results were analysed using the GeneMarker V2.2.0. software .A minimum of 50 ng of genomic DNA was isolated from each cell line for Short Tandem Repeat (STR) profiling. Briefly, the cells were harvested after reaching ~85% confluence. Genomic DNA was extracted from cell pellets using the DNeasy Blood & Tissue Kit  as per the manufacturer’s instructions. The samples were submitted to the Analytical Facility at the Queensland Institute of Medical Research  to establish the DNA profile. Nine STR loci , 10 μL/mL colcemid was added (Sigma Aldrich). This compound binds to tubulin, preventing the formation of mitotic spindles and thus helps to increase the number of cells in the metaphase state. The flasks were then submitted to Sullivan Nicolaides Pathology  for assessment. After disrupting the nuclear membrane, DNA was chemically fixed, and the structure and quantity of the chromosomes were analysed under a microscope and imaged by the pathology provider.https://www.atcc.org/). These cells were cultured as follows: RWPE-1, RWPE-2 and HPr-1 were cultured in KSFM containing Bovine Pituitary Extract  and epidermal growth factor . The remaining cell lines were cultured in phenol red free RPMI-1640 media containing glutamine (Glu+) + 10% FBS and 1% penicillin/streptomycin (ThermoFisher Scientific). All cell lines were maintained in a tissue culture incubator at 37 °C and 5% CO2. The cells were passaged upon reaching 70–80% confluence with 0.25% trypsin/EDTA . For morphology studies, the cells were seeded into 6-well plates and imaged using a Nikon Eclipse Ti microscope .Cell lines were cultured as noted above alongside a panel of two prostatic benign (RWPE-1 and HPr-1), one benign prostatic hyperplasia (BPH-1) and one prostate stromal (WPMY-1) as well as four prostate cancer cell lines  in parallel as references. All commercial cell lines were obtained from the American Type Culture Collection  + 10% FBS in a 96-well clear bottom black plate . One plate for each time point was seeded and incubated at 37 °C for 96 hours. Every 24 hours, one plate was removed from the incubator and treated with 132 µL/well of CyQuant dye, following the manufacturer’s instructions. Fluorescence (520 nm) was then measured after excitation (480 nm) using a microplate reader PolarSTAR Optima (BMG Labtech). Eight technical replicates were included for every cell line and the experiment was performed independently three times.Cell proliferation was measured using the CyQuant assay , which quantifies nuclear cell DNA content as a direct representation of the total cell number. For this, 2000 cells/well were cultured in 100 µL RPMI-1640 (Glu+) + 10% FBS for a final 0.5% agar concentration and a total of 2 mL per well was added to a 6-well plate . The agar was left for 15 minutes at room temperature to solidify before adding the top layer consisting of 0.66% Ultra-pure LMP agarose dissolved in PBS. An equal amount of media containing a single-cell suspension was immediately and carefully dispensed on top of the agar layer. After one hour incubation, allowing the top layer to solidify at room temperature, the plates were incubated at 37 °C. The new prostate cell lines were seeded at 20,000 cells/well while the controls (RWPE-2 and PC-3) were seeded at a final concentration of 10,000 cells/well. Different cell densities were used as starting points to account for the different cell growth rates observed in the proliferation assays. After three weeks at 37 °C, the colonies were pre-washed with PBS and stained with 0.5 mL fresh 0.01% crystal violet (Sigma-Aldrich) for 15 min at room temperature. Colonies were imaged with a Nikon Eclipse Ti microscope . Three technical replicates included in two independent experiments.The anchorage independent growth assay was performed for all four cell lines  as well as RWPE-2 and PC-3 as positive controls. Briefly, the bottom layer was prepared with 1% (w/v) Ultra-pure LMP agarose  dissolved in PBS and cooled to 40 °C. The agarose solution was diluted with RPMI-1640  were assessed for AR expression using immunofluorescence. Each cell line was plated at 60% confluence onto 6-well plates in triplicate, followed by fixing with 4% paraformaldehyde (Sigma-Aldrich) for 10–15 minutes at room temperature. Cells were incubated for 10 minutes in 0.25% Triton X-100 (Sigma-Aldrich) followed by three washes with PBS (5 minutes each). The fixed cells were incubated for 30 min in 2% BSA in PBS-Tween20 (BSA/PBST) solution followed by an overnight incubation with the primary AR antibody  at 4 °C on an orbital shaker. After three five-minute washes with PBS, the cells were incubated with Alexa Fluor 488 labelled secondary antibody  at 1:500 dilution in 1% BSA/PBST for one hour at room temperature on an orbital shaker. Two washes with PBS, followed by a final wash with water, were performed prior to nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) and mounting in Prolong Gold .+) + 5% Charcoal Stripped Serum  for 48 hours prior to the assay. Cells were seeded at 900,000 cells/well in 6 well plates (2 mL/well) and grown for 48 hours at 37 °C. Cells were then washed with PBS and fresh media was added followed by the treatment: either vehicle (20% ethanol) or 10 nM DHT for an additional 24 hours. RNA was subsequently isolated using the RNeasy Mini Kit (QIAGEN) following the manufacturer’s instructions.To confirm the expression of an active form of AR, the four new cell lines were treated with vehicle (20% ethanol) or with androgen  as described previously . The AR-AR: forward 5’-AAAAGAGCCGCTGAAGGGAA-3’, reverse 5’-GAAGACGACAAGATGGACAATTT-3’; KLK3  positive control: forward 5’-AGTGCGAGAAGCATTCCCAAC-3’, reverse 5’-CCAGCAAGATCACGCTTTTGTT-3’; RPL32 housekeeping gene: forward 5’-CCTTGTGAAGCCCAAGA-3’, reverse 5’-GACTGGTGCCGGATGAACTT-3’ (Sigma-Aldrich). The qRT-PCR analyses comprised of three biological replicates, unless stated otherwise, and three technical replicates per plate. The assay was performed on a ViiA 7 Real-Time PCR System .Total RNA was reverse transcribed into cDNA using the SensiFAST cDNA synthesis Kit (Bioline) as per manufacturer’s protocol. Quantitative reverse transcription PCR (qRT-PCR) was performed in a MicroAmp Optical 384-well plate  and the amplification was measured with SYBR Green . The primers used were as follows, A minimum of 10 µg of total RNA was extracted in duplicate from the newly established  and commercial  prostate cell lines using the RNEasy Plus Mini kit (QIAGEN) as above. Briefly, cells were grown in T75 flasks  until they reached 80% confluency. The extracted RNA was quantified using the NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific) and RNA integrity was assessed by Bioanalyser RNA Analysis (Agilent Technologies). All samples had a RIN value of 10, indicating an optimal sample quality.RNA-seq library preparation and transcriptome sequencing of the ten prostate cell lines was performed at the Garvan Institute of Medical Research . RNA sequencing libraries were prepared using the Illumina TruSeq RNA Sample prep Kit . Library preparations were sequenced on an Illumina HiSeq 2000 sequencer (Illumina) and 125 bp/150 bp paired-end reads generated using. RNA sequencing reads were mapped against the human genome . Samples with no counts were assigned a log2 CPM values of -5. Count data and associated scripts are available at https://github.com/sciseim/onyvax_MS.Read counts were obtained from TopHat-generated BAM files using SAMtools v0.1.19  and raw https://github.com/sciseim/onyvax_MS.Generalized fold change (GFOLD) v1.1.2  was emplHerein, we present four immortalised cell lines derived from the primary adenocarcinomas or adjacent benign tissue from four prostate cancer patients aged 59–69 years. All four patients presented essentially low to moderate disease  commercially available prostate cell lines was performed using RNA-seq data. RNA-seq produced approximately 35–43 million reads, with 51–59% of reads mapping to an annotated gene  are observed in LNCaP , while the other cell lines have close to zero counts.We performed the same analysis in the context of genes associated with prostate cancer tumorigenesis by assessing the prostate cancer pathway gene expression in the Kyoto Encyclopedia of Genes and Genomes , 31 (mapKLK3 (PSA) gene expression in prostate-derived cells. Due to the significant role that AR and androgens play in prostate cancer initiation and development, AR expression and androgen response assays were performed. IF analysis on the two cancerous cell lines shows that AQ0411 has a low AR expression while AQ0415 shows none  were within the following ranges: (∆Ct)vehicle = 15.7–17.30 and (∆Ct)DHT = 15.99–17.48, while values for the positive LNCaP control are (∆Ct)vehicle = 4.7–5.2 and (∆Ct)DHT = 10.7–8.A functional androgen receptor would be expected to drive ent Fig. . The posCD44) and basal  markers assessed. Expression of additional stem cell markers, a2-integrin (CD49B) and BCRP (ABCG2) was lower in the metastatic LNCaP, and PC-3 cells and higher expression observed for the four new cell lines (Supplementary Table PSCA) was positive in AQ0396, AQ0420, AQ0411, LNCaP and RWPE-2. AR and KLK3/PSA are highly expressed in LNCaP as expected. AR expression in AQ0396 was detected, but this was comparatively very low: an RPKM of 0.77 compared to 67.5 in LNCaP. Only LNCaP and the benign AQ0396 showed KLK2 expression. Also of note, although only the benign cell line AQ0420 expresses ERG, several other key genes expressed in the prostate and during tumorigenesis – AMACR, GSTP1, NOTCH1, PCNT1, PTEN and NES – are expressed in all four newly derived cell lines.To confirm the presence of heterogeneous cell morphology typical of prostate cancer disease , 33, marSince their establishment many decades ago, the most commonly used cell lines for prostate cancer research are all derived from metastases: LNCaP from the lymph node , PC-3 frThis study has characterised cell lines, derived from radical prostatectomy specimens obtained from four men with localised prostate adenocarcinoma, in monolayer cell cultures. All cell lines exhibit an epithelial cell morphology and proliferate at similar rates. Chromosomal ploidy imaging shows inter- and intra-tumour heterogeneity in both of the tumorigenic cell lines, typical of many cancers , includiITGB1, CAV1 and low expression of CRYBA1, CEACAM5 were observed in all, suggesting the lack of anchorage-independent growth of the two cancer-derived cells is due to different mechanisms than lack of anoikis resistance. Moreover, proliferation may be influenced by other factors such as seeding density that determines the cell-to-cell contacts, affects proliferative ability of the cell lines and the selection of subclones during passaging.Neither of the two cancerous cell lines can form colonies in an anchorage-independent manner, despite this process being known to be a tumorigenesis cell hallmark . We initPSCA expression. This marker is highly expressed in prostatic tissue compared to other tissues [PSCA has been extensively studied in high-grade and later stages of the disease [PSCA in these early stages. On this note, a study comparing data sets from the tumour and adjacent non-cancerous and healthy tissues from eight cancers (including prostate cancer) found that shared signalling pathways across tissues were activated in the adjacent non-cancerous cells, proving molecular changes associated with a pre-cancerous stage [A heterogeneous prostatic lineage, typical of the early stages of prostate cancer disease  is obser tissues , 52. Whi disease –56, what disease , need tous stage .AR reported isoforms, improving our understanding of their roles in tumour initiation and development. Furthermore, AR expression declines after cell passaging [AR expression/isoforms may help elucidate the AR-dependent molecular mechanisms that contribute to the initiation and development of the disease. Interestingly, in a study where samples from primary prostate cancer and benign prostatic hyperplasia were compared, three AR isoforms were dysregulated, and their opposing expression profiles correlated with disease aggressiveness [AR, and other genes, when growing under different conditions than a monolayer. In this study, we have measured AR-response by expression analysis of PSA/KLK3. However, PSA expression is critically dependent on cell morphology. PSA expression is observed in tall columnar cells of the prostate gland and often high in cells which are larger and well differentiated [None of the four cell lines is AR-responsive after androgen treatment and no expression was observed neither by immunofluorescence nor RNA-seq, with the exception of the cancerous AQ0411 and benign AQ0396 cells. AQ0411 cell line exhibited low AR expression only in our immunofluorescence assays, while AQ0396 showed low AR expression in the RNA-seq analysis. These discrepancies are likely due to the polyclonal origin of this cell line, or driven by HPV integration as a result of the HPV16 E6/E7 immortalization process, one of the standard method for immortalization, at the time when these new cell lines were established , 59. Theassaging  and the assaging –4, expreassaging . None ofassaging . The comassaging  originatsiveness . Howeverentiated . We obseentiated –68. Monoentiated –71. Usinentiated , 73. TheTo conclude, the cell lines characterised in this study derived from localised prostate adenocarcinoma (AQ0411 and AQ0415) and adjacent benign-tissue (AQ0396 and AQ0420), have the potential to assist to further broaden research on early-stage prostate cancer and its impact on the surrounding benign prostate. Indeed, our preliminary data suggests these cell lines have the potential to further delineate between indolent and what may evolve towards clinically significant disease. This will require further analysis to determine which clinical states are most closely represented by these cell lines and whether more complex culture conditions such as 3-dimensional approaches or co-culture with other relevant cell types will also be useful. Nevertheless, this study has several limitations, such as the effect of the cell passage number on the genetic and molecular characteristics of the cell lines, or lack of data showing their ability to form tumours in xenograft mice models. We intend to deposit these cell lines in a publicly accessible Authenticated Cell Repository so additional investigation of these cell lines in 3D spheroids and pre-clinical models can be assessed and their usefulness as models of early prostate cancer can be further elucidated.Additional Supplementary MaterialSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Figure 1Supplementary Figure 2


Soil pollution is a global environmental problem. Nanoscale zero-valent iron (nZVI) as a kind of emerging remedial material is used for contaminated soil, which can quickly and effectively degrade and remove pollutants such as organic halides, nitrates and heavy metals in soil, respectively. However, nZVI and its composites can enter the soil environment in the application process, affect the physical and chemical properties of the soil, be absorbed by microorganisms and affect the growth and metabolism of microorganisms, thus affecting the ecological environment of the entire soil. Because of the potential risks of nZVI to the environment and ecosystems, this paper summarizes the current application of nZVI in the remediation of contaminated soil environments, summarizes the various factors affecting the toxic effects of nZVI particles and comprehensively analyzes the toxic effects of nZVI on microorganisms, toxic mechanisms and cell defense behaviors to provide a theoretical reference for subsequent biosafety research on nZVI. Nanomaterials are an important development in nanoscience and technology, and can be defined as any organic, inorganic or organometallic material that is in at least one dimension of 100 nm in three dimensions and exhibits chemical, physical and electrical properties that vary with the size and shape of the material . Thus, t2+, Cd2+, Cu2+ and Ni2+ from soil, and with the increase of nZVI concentration, the adsorption of all metals increased, and the adsorption of Pb2+ and Ni2+ reached almost 100% [6+ in soil into Cr3+, thereby reducing the toxicity to the soil [Escherichia coli and Bacillus subtilis in soil. Chaithawiwat et al. [E. coli after nZVI staining at 10–70 nm particle size and 1000 mg/L concentration. The results showed that the toxicity of nZVI was more serious during the exponential growth phase and decay phase of E. coli, and the toxic effects were strain-dependent. In general, the toxicity of nZVI was positively correlated with its concentration. However, nZVI has also been shown to exhibit stronger toxicity to Agrobacterium sp. PH-08 at low concentrations after 2 h of contact. [Nanoscale zero-valent iron (nZVI) refers to zero-valent iron particles with a particle size of 1–100 nm, which have a large specific surface area, strong reduction, strong adsorption, and a unique oxide shell compared to bulk materials . These post 100% . In addithe soil ,12,13. Athe soil ,15. Micrthe soil  studied t et al.  studied contact. . Therefocontact. ,20.Although some studies have described the toxicological effects of nZVI on plant cells , animal With the development of urban industrialization and intensive agriculture in various countries around the world, organic and inorganic contaminants  are spread into the soil without treatment or improper treatment. Soil pollution is insidious and long-term, and toxic and harmful substances will have a serious impact on the ecosystem after infiltrating into the soil through the material cycle . A large2O2, 29.88 g/kg MMT-nZVI and an initial pH of 3.5. DBDPE is the main material for the production of electronic products, plastics and textiles, and its unique degradability and thermal stability make DBDPE more likely to accumulate in soil [3− by chemical reduction in soil remediation, but can also act as an electron donor in combination with microorganisms or coupled with bioelectrochemical system to degrade nitrate by the denitrification process [2 group, followed by a dechlorination reaction. The degradation rate of 0.3 mM CAP in the first stage  was up to 97%, and the reaction resulted in a great reduction in CAP concentration.Halogenated organic pollutants mainly include fluorinated, chlorinated and brominated pollutants, which are highly persistent and difficult to biodegrade, and also have toxic characteristics and can be a serious ecological hazard if they enter the environment. Stabilized nZVI has been commonly studied for the reductive dechlorination of chlorinated organic pollutants. Polychlorinated biphenyls (PCBs) are widely used in industrial production. Due to their high hydrophobicity and volatility, the natural attenuation of PCBs in soil is usually very slow and tends to accumulate in organic matter-rich soils ,26. Sun  in soil . Lu et a in soil  loaded n process . The rea process . Liu et  process  degradedα-Proteobacteria and β-Proteobacteria. Fajardo et al. [β-Proteobacteria subclass increased to 21.8%, while in the ε-Proteobacteria subclass, the percentage decreased to 6.1%. Vanzetto et al. [Bacillus cereus and Pseudomonas aeruginosa colony-forming units (CFU) in hexavalent chromium (Cr6+) and pentachlorophenol (PCP) nanoremediation contaminated soil. The results showed that nZVI in Cr6+ and PCP-contaminated soil had no toxic effect on the population of native soil bacteria. At 90 days after nZVI injection, the mean value of CFU was statistically equal, with the lowest coefficient of variation and the highest level of CFU. This is because the strains of B. cereus and P. aeruginosa are resistant to the concentrations of nZVI, Cr6+ and PCP, and did not cause significant disturbances in temperature, conductivity, pH and humidity over time. Furthermore, nZVI is not solely toxic to microorganisms in soil aquifers. It has been shown that nZVI inhibits the physiological activity of some microbial groups  but promotes the growth of others  [Soil is an interface matrix between different substances  and organisms, with multiple layers and complexity. The unique reaction characteristics of nZVI particles with pollutants in soil ensure the advantages of nZVI in situ remediation of contaminated soil. o et al.  found tho et al.  evaluateacteria) ,39. The acteria) ,40,41.Microorganisms play a very important role in the ecological environment. As active components in soil, microorganisms not only regulate the organic matter, nutrient cycling and energy conversion, but also are sensitive to microenvironmental changes. In addition, their microbial activity and community structure changes are sensitive indicators of soil quality and health. Microorganisms play a key role in ecosystem function and productivity, and the toxicity of nZVI to microorganisms has received widespread attention ,39. As sE. coli under anaerobic conditions than under aerobic conditions [Bacillus subtilis, while nZVI has a good inactivation effect on Gram-negative bacteria such as Escherichia coli [The size and dose of nZVI have a significant effect on its toxicity. Generally, the particle size decreases and the toxicity increases. For example, the toxicity of nano-zero-valent iron is greater than that of micro-zero-valent iron . Howevernditions ,44. nZVInditions . The toxhia coli . The surhia coli . Table 2B. cereus was less than 1000 mg/L after 2 h, and significant toxicity was still observed after 24 h, but only at the lowest concentration [Pseudomonas stutzeri was inhibited at the initial stage of contact with nZVI, and then the growth gradually returned to normal. Similarly, highly oxidized (mature) nanoscale zero-valent iron particles were less harmful to the microbial population compared to fresh nZVI [P. stutzeri and nZVI. It may be due to the fact that nZVI polymerizes and settles or gradually oxidizes with time, thus reducing nZVI toxicity. Interestingly, Gonzalo et al. [In general, the toxic effects of nZVI on bacterial species tend to increase with the increase in nZVI concentration . Howeverntration . This isntration  found thesh nZVI . Saccà eesh nZVI  confirmeo et al.  found a In the past decade, while the toxic effects of nZVI on microorganisms have been revealed, there have been many studies on the mechanisms of its toxicity, but no uniform conclusion has been reached. According to the literature, the possible mechanisms of nZVI toxicity include the following four types: morphological or functional damage of microbial cell membranes, oxidative damage, release of iron ions and genetic damage see .nZVI can simply adhere to microorganisms by virtue of its physicochemical properties, such as Brownian motion, electrostatic attraction, Coulomb attraction, hydrogen bonding and even dipole-dipole collaboration. The adsorption of nZVI on microbial cells leads to direct contact with cell membranes. Lv  found th2) to form reactive oxygen species (ROS), which mainly include O2, O2−, H2O2 and ·OH. This type of excessive reactive oxygen species cannot be removed in time by the intracellular antioxidant defense system, so they can accumulate in large amounts in cells, resulting in an imbalance between intracellular oxidation and antioxidant status. This results in oxidative stress, which causes macromolecules such as lipids, proteins and nucleic acids to denature, damaging cell structure and eventually causing cell death. [E. coli strains and E. coli mutant strains lacking antioxidant genes to nZVI, respectively. The results showed that mutant strains were more vulnerable to damage, which confirmed that oxidative damage was one of the toxic mechanisms of nZVI.Reactive oxygen species generation and oxidative stress reactions are currently recognized by academia as the main ways in which nanomaterials cause biotoxicity . Due to l death.  At the sl death. . Krittanl death.  exposed 0 as the core being wrapped by iron oxide in the outer layer. Once exposed to the external environment, the thickness of the external oxide layer of the nanoparticles increases rapidly [2+ and Fe3+ are continuously released. Trace amounts of Fe ions are indispensable for the growth of living organisms, but when there is an excess of Fe ions, it can be harmful to the organism. The reaction of releasing iron ions until the zero-valent iron core is completely oxidized, and the relevant equation is as follows [At the initial stage of cell exposure to nZVI, nZVI is rapidly oxidized, resulting in nZVI with Fe rapidly . Fe2+ an follows :Fe0+2H2O2+ is preferentially released on the surface of nZVI relative to Fe3+, the former being released on the surface of nZVI. In contrast, the strong reducing nature of nZVI decreases the permeability of the cell membrane and facilitates the entry of iron ions into the cell to react with H2O2 to generate excess ROS, causing oxidative stress and subsequent cell death [2+ and Fe3+ in the intracellular Fenton reaction are as follows:Normally, Fell death . The equ2+ and Fe3+ are inevitably and gradually transformed into iron oxides in the environment. Qiu et al. [Feu et al.  exposed With the increasing application of nanomaterials in the engineering field, there is also a growing interest in the impact of nZVI at the genetic level. Gomes et al.  examined3+ , Hg2+ and Pb2+, and further exhibit toxicity in concert with these heavy metals [In fact, the toxicogenic mechanism of nZVI may not be unique, but may be a combination of multiple mechanisms. In addition to the four points mentioned above, the mechanism of toxicity may also be that nZVI has strong adsorption properties and will actively adsorb toxic and harmful substances in the environment, such as thr heavy metals Asy metals ,72. In oP. stutzeri, which plays an important role in nutrition and iron absorption, and that a decrease in protein abundance can reduce iron availability intracellularly and cause intracellular oxidative damage. A unique defense measure was found in Bacillus cereus, a common Gram-positive bacterium in soil. When Bacillus cereus is exposed to nZVI particles, asymmetric septa are formed in the cell, which are typically in the early spore-forming phase, so that it can survive the stressful environment and regulate the expression rate of specific proteins [K. oxytoca may establish an adaptive stress response, including indole and tryptophanase, which can be regarded as signal molecules. The overexpression of tryptophanase may indicate that the protein is involved in the cell adaptation process of K. oxytoca, which can reduce or even offset the negative effects of nZVI. In addition, the activities of some common intracellular antioxidants, such as glutathione reductase (GR), superoxide dismutase (SOD) and catalase (CAT), also change in response to elevated intracellular ROS levels. For example, catalase, encoded by the katB gene, is involved in cellular oxidative stress detoxification. Upregulation of katB occurred after exposure of p.stutzeri to nZVI. This phenomenon indicates involvement of katB in H2O2-induced oxidative stress. The mechanism of microbial toxicity when exposed to nZVI was proposed, as shown in The mechanistic response of bacterial or fungal cells to resistance induced by nZVI toxicity is being elucidated. It has been shown in the literature that changes in protein expression indicate that cells trigger a molecular response to counteract the effects of nZVI through two main mechanisms: inhibition of iron uptake by membrane proteins; and overproduction of proteins to scavenge ROS, thereby reducing intracellular oxidative stress . Saccà eproteins . Accordiproteins , K. oxytAs mentioned above, the factors that influence nZVI toxicity are complex. Although most toxicity studies have been conducted with bare nZVI, nZVI particles used for in situ remediation are usually surface-stabilized, and the type of stabilizer used not only increases the mobility of nZVI but also attenuates the toxicity of nZVI. Currently, surface modification is used to improve the stability of nZVI due to the limited nature of nZVI itself. At the same time, the only artificially controllable factor to reduce the toxicity of nZVI is the modification method of nZVI. The selection and preparation of modified materials have an impact on the toxicity of nZVI, and the selection of green synthetic modified coating materials can effectively mitigate the toxicity of nZVI. Various types of surface modifiers/stabilizers, such as surfactants, polymeric organics and polyelectrolytes, are used as coating materials to reduce agglomeration/rusting and improve the flowability of nZVI. These stabilizers can provide stability to nanoparticles by applying stabilizing forces  and spatial or even electrospatial stabilization between nanoparticles. The electrostatic repulsion  provided by the large molecular weight polyelectrolyte significantly inhibits the physical contact between nZVI and the bacterial cell wall in the stabilization of nano-zero-valent iron by the spatial repulsion promoted by the negatively charged polymer coating. Thus, it reduces the toxicity of nZVI to microorganisms . In addiAs an emerging material for environmental remediation, the negative effects of nZVI on the environment need to be focused on. However, the toxic effects and influencing factors of nZVI in the environment are complex, and the current characterization methods are very limited, so our knowledge of nZVI is still in the initial stage. Studies on the toxic effects of nZVI are just beginning at this stage, and not much research has been conducted in real environments. There are many challenges for future toxicological studies on nZVI on soil microorganisms.(1)The factors affecting the toxicity of nZVI to soil microorganisms are complex and are influenced by the nature of nZVI itself, whether it is modified, the amount used, and even in practical applications, the soil properties and the linkage with other substances in the environment. In addition, the result of the interaction between nZVI and other substances in the soil in practical application may enhance the toxicity of nZVI or weaken it, and strengthening the research in this aspect is beneficial to determine the performance of nZVI in the actual environment.(2)The mechanisms of nZVI toxicity are currently agreed upon by academics as morphological or functional damage to microbial cell membranes, oxidative damage, release of iron ions and genetic damage. The first three are toxicological studies at the cellular level, while gene damage is a study at the molecular level and is still in the research stage. Although there are many hypotheses about the mechanism of toxicity, the exact mechanism is still unclear, and the existence of other mechanisms needs to be further investigated, taking into account environmental factors, test organisms and the properties of nZVI itself.(3)The defenses of microbial cells in soil against nZVI toxicity are currently more clearly elucidated at the cellular level, but studies at the genetic level are still lacking. Meanwhile, the commonalities and characteristics of cellular defense behaviors need to be further investigated and summarized.(4)Toxicity studies on the temporal effects of nZVI need to be strengthened. nZVI toxicity may be a long-term and chronic process after injection into the environment, and under certain conditions, the short-term toxic effects may not be obvious, so there is a need to strengthen the long-term and large-scale studies on the toxic effects of nZVI on the ecological environment.


Despite strong epidemiological evidence that dietary factors modulate cancer risk, cancer control through dietary intervention has been a largely intractable goal for over sixty years. The effect of tumour genotype on synergy is largely unexplored.® assay. Synergy, additivity and antagonism were modelled using Bliss additivism and highest single agent equations. Patterns of maximum synergy were identified by polygonogram analysis. Network pharmacology approaches were used to identify interactions with known PCa protein targets.The effect of seven dietary phytochemicals, quercetin (0–100 μM), curcumin (0–80 μM), genistein, indole-3-carbinol (I3C), equol, resveratrol and epigallocatechin gallate (EGCG) (each 0–200 μM), alone and in all paired combinations om cell viability of the androgen-responsive, pTEN-null (LNCaP), androgen-independent, pTEN-null (PC-3) or androgen-independent, pTEN-positive (DU145) prostate cancer (PCa) cell lines was determined using a high throughput alamarBlueSynergy was observed with all combinations. In LNCaP and PC-3 cells, I3C mediated maximum synergy with five phytochemicals, while genistein was maximally synergistic with EGCG. In contrast, DU145 cells showed resveratrol-mediated maximum synergy with equol, EGCG and genistein, with I3C mediating maximum synergy with only quercetin and curcumin. Knockdown of pTEN expression in DU145 cells abrogated the synergistic effect of resveratrol without affecting the synergy profile of I3C and quercetin.Our study identifies patterns of synergy that are dependent on tumour cell genotype and are independent of androgen signaling but are dependent on pTEN. Despite evident cell-type specificity in both maximally-synergistic combinations and the pathways that phytochemicals modulate, these combinations interact with similar prostate cancer protein targets. Here, we identify an approach that, when coupled with advanced data analysis methods, may suggest optimal dietary phytochemical combinations for individual consumption based on tumour molecular profile. In histsorption . Single patients , single r (CRPC) . These fr (CRPC) , 8. A rar (CRPC) . Other cr (CRPC) , 11. Majr (CRPC) , 13. Basr (CRPC) , we testr (CRPC) , quercetr (CRPC) , indole-r (CRPC) , equol (r (CRPC) , curcumir (CRPC) , resverar (CRPC)  and EGCGr (CRPC) . When co2.Cell lines  were obtained from the ATCC  and identity was confirmed by short tandem repeat analysis (data not shown). Relevant characteristics of cell lines –33 are sQuercetin and I3C were purchased from Sigma Aldrich  genistein, EGCG, curcumin, RS-equol, and resveratrol from Tocris Bioscience . All compounds were > 95% purity as supplied. Compound stock solutions (10 mM in dimethyl sulfoxide (DMSO) (Sigma Aldrich)) were stored at-30°C prior to use.® assay configured for high throughput analysis) were performed in duplicate experiments . Cells in logarithmic phase growth were seeded ) onto black, 384 well plates  and were exposed immediately to test phytochemicals or assay controls , negative control, DMSO) either by use of assay ready plates or robotic compound addition. All assays contained DMSO at 0.2%. A 10-point serial dilution of stock solutions was prepared in 384 well polypropylene plates  and dispensed to assay plates  equipped with a 384-head pin tool ). Metabolic activity was detected 72 h post-phytochemical exposure by the addition of alamarBlue reagent  (Multidrop Combi reagent dispenser (Thermo Scientific)) and fluorescence intensity determination .Cells were maintained as adherent cultures  in RPMI medium  supplemented with 10% foetal bovine serum, (Life Technologies). Cell viability assays ^C) (Eq. 2) where A = baseline response parameter, B = maximum response parameter and C = Hillslope of the dose–response curve respectively, Pair-wise combination studies were conducted using a six-point, two-fold dilution series centred on the IC50 concentration, where possible, in a 36-point dose matrix. Compound dilution stocks were pre-dispensed serially to assay plates . Subsequently, cells were seeded onto these assay-ready screening plates for cell viability assays. Final concentrations were: genistein, equol, I3C and resveratrol (for LNCaP and PC-3 cell lines) and EGCG, 10, 25, 50, 100, 200 μM; quercetin: 5, 12.5, 25, 50, 100 μM; curcumin: 4, 10, 20, 40, 80 μM; resveratrol (DU145): 2, 5, 10, 20, 40 μM. Synergy analysis was conducted using the BLISS and highest single agent (HSA) analysis methods (Cell viability relative to positive (pos) and negative (neg) assay controls were calculated from relative fluorescence unit (RFU) data as follows: % viability = (RFUsamples-RFUpos) (RFUneg-RFUpos) × 100 (Eq. 1). Positive control experiments showed complete suppression of fluorescence (data not shown). IC50s  were cal methods , 35  according to the manufacturer’s protocol. Assay absorbance was read at 490 nm on a spectrophotometer .pTEN knockdown experiments were performed on DU145 cells using pTEN siRNA cocktail oligonucleotides (cat no: AM51331) and control siRNA oligonucleotides at a dose of 20 nM . The siRNAs were transfected into cells with Lipofectamine 2000  in a serum-free medium according to the manufacturer’s instructions. Following 48 h transfection, cells were treated with respective phytochemical combinations for 72 h. Cell viability was then assessed using a CellTiter 96Western blot analysis was performed as described previously . Brieflyp-values were adjusted for multiple hypothesis tests using Benjamini and Hochberg False Discovery Rate (FDR) correction. The KEGG pathways with FDR < 0.05 were regarded as significant and reported. A comprehensive platform of gene-disease associations, MalaCards  , and DrualaCards , was useTo investigate the pharmacological actions of the synergistic phytochemicals, networks showing the interactions among phytochemical compound combinations, protein targets, biochemical pathways and “causal” prostate cancer genes were constructed. Protein–protein interactions (PPIs) among targets and PCa-related genes were obtained from the comprehensive resource of validated and high-confidence predicted PPIs, “Interologous Interaction Database,” I2D version 2.9 . The net3.50 9 to 12 μM) (50 < 25 μM). DU145 cells were the most sensitive (5/7 compounds IC50 < 100 μM).Curcumin was the most potent single agent in all cell lines (apparent ICo 12 μM) . ResveraDual phytochemical treatment analysis by Bliss or HSA models showed cell-line-dependent responses . The mosThe HSA model accentuates synergistic responses, while the Bliss model accentuates antagonism at high concentrations of curcumin and I3C. For combinations with relatively weak synergy or antagonism, the two models give different results. The two models gave inconsistent synergy results for resveratrol/genistein (LNCaP) , combinaHigher-order relationships between maximally synergistic phytochemical combinations (Bliss results) were identified using polygonograms  Figure . These r50 < 4.5 μM quercetin with 100 μM I3C) and in PC-3 cells (IC50 < 4.5 μM quercetin with 200 μM I3C). In contrast, despite displaying lower overall synergy than I3C/quercetin (50 ~ 2.8 μM resveratrol with 25 μM equol). Overall, the greatest synergistic impact on potency in combination with other phytochemicals with 15/21 (71%) maximally effective combinations involved I3C. I3C with quercetin or curcumin was synergistic in all cell lines. In combination, I3C/curcumin showed greater potency than curcumin alone, with IC50 of the combination improving by ~10-fold in LNCaP cells and 2-fold in PC-3 and DU145 cells. Our synergy data predicts that the pattern of the maximum synergy of dual combinations of each of the seven phytochemicals is dependent on cellular genotype. Our hypothesis was that pTEN loss, a common feature in castrate-resistant prostate cancer patients and seen in AR-positive LNCaP and AR-negative PC-3 cells, reduces the influence of resveratrol on maximum synergy in DU145 cells. To test this, pTEN knockdown experiments were performed in DU145 cells  shared direct interactions with I3C, which also linked to 10 additional proteins . PathwayNetwork pharmacology analysis of equol/resveratrol, the most effective DU145 combination , identifThe effect of the major genotypic alterations in each of the three cell lines LNCaP, , PC-3 , and DU145  on the PCa protein interactome for each cell line was also examined . pTEN lo4.I3C is identified here as a major promoter of synergy featuring in 12/18 (67%) maximally synergistic combinations . I3C is in vitro and achievable concentrations in vivo that is seen with some, but not all, combinations.Every compound showed synergy with multiple phytochemicals; however, the potency of cell killing with maximally synergistic combinations varied significantly for each phytochemical and between cell lines . In DU14Pattern analysis revealed a strikingly similar profile in pTEN-null LNCaP and PC-3 cell lines, despite their dramatically different androgen responsiveness, in contrast to pTEN-positive DU145 cells . Thus, tIn contrast to AR status, responsiveness appears to reflect the pTEN status of the cell lines. The results of our pTEN knockdown experiments in DU145 cells  confirm in vitro benefit and the imprecision of our understanding of the genetic targets of common phytochemicals. The finding here, that a limited number of genes critical for genome integrity may be the targets of phytochemical treatment could focus research in the field and provide potential pathways to study patients showing pre-neoplastic lesions that often precede the onset of tumorigenesis.Our network pharmacology approach suggests that, despite individual phytochemicals having diverse interactomes, the most potent phytochemical combinations target a similar subset of proteins known to be associated with PCa progression  and thatIn contrast to the equol/resveratrol combination, the knockdown of pTEN was insufficient to improve the potency of the I3C/quercetin combination in DU145 cells at concentrations that are maximally synergistic in PC-3 cells . This suImportantly, we have shown that the variation in individual synergistic response is a genetic trait. Again, to our knowledge, this has not been shown earlier and has potentially great promise in understanding how synergy and antagonism occur. Specifically, if there is a genetic basis for phytochemical synergy, this could explain why individual responses to dietary intervention vary from person to person. Maximally synergistic phytochemical concentrations approached physiologically relevant concentrations equating to dietary intake. Therefore, our study may also be helpful in defining a diet of whole foods based on phytochemical concentrations. A 30-day intake of 4 g/d curcumin resulted in blood levels up to 11.9 μM , while 3viz. the prediction of the most effective combination of phytochemicals to include in a patient dietary plan based on a genetic, transcriptomic, proteomic and lipidomic molecular profile of a patient tumour taken at biopsy or on surgery. The approach may also be useful over the course of patient treatment by providing molecular-profile-guided dietary advice derived from the analysis of liquid biopsies.Application of high throughput approaches has its limitations in that the number of experiments needed to determine synergy grows exponentially with the number of combinations screened. Recent advances in mathematical modelling have identified a way of predicting the effect of multiple combinations of drugs based on measurements of drug pairs , thus re5.In summary, our findings provide a systematic analysis of synergy in three genotypically distinct prostate cancer cell lines, demonstrating that optimal phytochemical combinations are dependent on the molecular profile of tumour cells and provide a pathway for the development of patient-specific dietary advice based on tumour molecular profiling.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.CG, PdS, FV, and KS: conceptualization. SF, TF, and GA: methodology. SF and FV: software. SF, TF, GA, and MS: validation. TF, GA, SF, and FV: formal analysis. CG, SF, and KS: investigation. TF, GA, FV, DM, PdS, and KS: resources. TF, GA, SF, FV, and KS: data curation. CG, SF, TF, GA, DM, AS, SF, FV, and KS: writing – original draft preparation. CG, SF, AF, GA, MS, DM, AS, JC, JB, PdS, FV, and KS: writing – review and editing. CG, TF, GA, SF, FV, and KS: visualization. DM, JC, JB, PdS, and KS: supervision. CG and PdS: project administration. JB, DM, and PdS: funding acquisition. All authors contributed to the article and approved the submitted version.This project was supported by an Australian Research Council (ARC) PhD Scholarship award to CG.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Colorectal cancer (CRC) is the second-leading cause of cancer death, with a worldwide incidence rate constantly increasing; thus, new strategies for its prevention or treatment are needed. Here, we describe the adjuvant effect of the polyphenol-enriched fractions of cinnamon, from cinnamon bark and buds, when co-administered with a potent anticancer drug, cetuximab, used for CRC therapy. The co-administration significantly reduces the cetuximab dose required for the antiproliferative activity against colorectal cancer cell line E705, which is sensitive to EGFR-targeted therapy. The anticancer activity of these cinnamon-derived fractions, whose major components  are procyanidins and other flavonoids, strictly correlates with their ability to induce apoptosis in cancer cell lines through ERK activation and the mitochondrial membrane potential impairment. Due to the severe side effects of cetuximab administration, our results suggest the use of nutraceuticals based on the polyphenolic fractions of cinnamon extracts as adjuvants in the therapy of CRC. Colorectal cancer (CRC) is the second-leading cause of cancer death and the third-most prevalent malignant tumor worldwide; its incidence rate is constantly increasing . New appThe ideal CRC treatment would achieve complete removal of the tumor and metastasis, which mostly requires surgical intervention . For thoThe idea of molecular-targeted therapy has emerged in the last two decades. The epidermal growth factor receptor (EGFR) is a driver in many cancers and, as a consequence, is a major target in oncology. Monoclonal antibodies (mAbs), as well as small molecules like tyrosine kinase inhibitors, are used as a treatment for patients with a variety of solid tumors ,5. RegarTherefore, there is an urgent need to identify new approaches, such as the application of nutraceuticals, which are able to both elicit and expand the range of chemo-preventive actions while reducing the amount of drugs administered during a therapeutic cycle . NutritiCinnamon is a popular flavoring ingredient, widely used in food products. It exhibits health-beneficial properties; it has been widely used for treating blood circulation disturbances, dyspepsia, allergic disease, gastritis, diabetes, and hypertension  and has Cinnamomum cassia (CCHE) or Cinnamomum zeylanicum (CZHE) or buds from Cinnamomum cassia (BCHE)—and of the corresponding fractions enriched in polyphenols (B) or cinnamaldehyde (C)—on colorectal healthy and cancer cell lines have been evaluated. In particular, cell lines were treated for 48 h, and cell viability, apoptotic rate, and levels of P-ERK, caspase 3, and BCL2 were evaluated. These results allowed the elucidation of the anticancer activity and the potential molecular mechanism of cinnamon fractions enriched in polyphenols on human colorectal cancer cell lines.In the present study, the effects of hydroalcoholic extracts of bark from Cinnamomum cassia (CCHE) or Cinnamomum zeylanicum (CZHE) or buds from Cinnamomum cassia (BCHE) cinnamon was already reported by our group  or Cinnamomum zeylanicum (CZHE) or buds from Cinnamomum cassia (BCHE). The chosen cell lines were a healthy colorectal mucosa CCD841 cell line and three colorectal cancer cell lines with peculiar molecular features. In particular, Caco-2 and E705 show no hyperactivating mutations in KRAS, NRAS, BRAF, and PIK3CA genes, with the E705 cell line carrying a silent mutation in the PIK3CA gene, whereas the SW480 cell line carries a hyperactivating mutation in exon 2 of the KRAS gene. Caco-2 and SW480 cell lines do not respond to cetuximab, while the E705 cell line is sensitive to cetuximab [We evaluated the effect on the viability of different colorectal cell lines of hydroalcoholic extracts and fractions enriched in either polyphenols (B) or cinnamaldehyde (C) of bark from etuximab . MTT assays  revealedThe CCHE total extract showed a significant dose-dependent cytotoxic effect on Caco-2 and E705 cell lines, starting from 100 μg/mL concentration, and on SW480, starting from 50 μg/mL A. The CZThe polyphenol-enriched fractions from CCHE, CZHE, and BCHE extracts showed a significant increase in cell viability at 10 μg/mL in all the cell lines B,D,F. ByNoteworthy, fractions B at all concentrations did not affect the healthy cell line, demonstrating a selectivity against colorectal cancer cell lines.Fraction C enriched in cinnamaldehyde showed no significant effect on cell viability in any line up to a concentration of 50 μg/mL. We observed a toxic effect only on Caco-2 and SW480 lines at 100 μg/mL .To evaluate the combined effect of cinnamon fraction B and cetuximab, we used the cetuximab-sensitive E705 cell line, and the cells were treated with different concentrations of cetuximab (0–100 μg/mL) and at a fixed concentration of cinnamon fraction B (50 μg/mL). As reported in Furthermore, the fraction enriched in polyphenols from cinnamon bud extract (BCHE) was more effective at all cetuximab doses than the other two fractions. This fraction at 50 μg/mL was more effective than cetuximab at 0.1 μg/mL, and the effect is the same as the one shown by the drug at concentrations between 0.5 and 1 μg/mL.To examine whether the decrease in cell viability was due to apoptosis, we performed a flow cytometric analysis for Annexin V-FITC and PI and found that fractions enriched in polyphenols obtained from CCHE, CZHE, and BCHE hydroalcoholic extracts at a dose of 50 μg/mL induced apoptosis in all colorectal cancer cell lines. As reported in A stronger effect of all three fractions was observed on E705 cells, where the late apoptotic cells are 56%, 44%, and 52% after treatment with CCHE fraction B, CZHE fraction B, and CCHE fraction B, respectively . As repoWestern blot analyses showed different molecular mechanisms of apoptosis. In Caco-2 cells, only the treatment with BCHE fraction B induced a weak ERK activation. CCHE and BCHE fractions B induced a decrease in anti-apoptotic BCL2 factor and caspase 3 cleavage and activation A.In E705 cells, only the treatment with BCHE fraction B induced ERK activation, but treatment with any fraction B induced a BCL2 decrease and caspase 3 activation B. In SW4m) is an important parameter of mitochondrial function and an indicator of cell health. Treatment with CCHE, CZHE, and BCHE fractions B induced a loss of DiOC6 fluorescence in all colorectal cancer cell lines, indicating disruption of the mitochondrial inner transmembrane potential  or Cinnamomum zeylanicum (CZHE) or buds from Cinnamomum cassia (BCHE) induced biologically opposite effects at different doses on the viability of healthy colorectal mucosa CCD841 cell line and colorectal cancer cell lines Caco-2, E705, and SW480. The term hormesis has long been used to describe the phenomenon where a specific chemical can induce a stimulatory or beneficial effect at low doses and an inhibitory or toxic effect at high doses [The hydroalcoholic extracts and fractions enriched in polyphenols (B) of bark from gh doses ,25. At hAlthough fractions B obtained from the three different cinnamon extracts showed different UPLC–HRMS profiles , procyanFractions B from both CCHE and BCHE extracts were found to be more cytotoxic when compared to their respective original extracts and also to the fraction B obtained from CZHE extract, suggesting a role in their antiproliferative activity either for B-type procyanidins or for flavonoid glycosides. Interestingly, fraction C, mainly containing cinnamaldehyde and related compounds, when tested at the same concentrations of fractions B, was not found to be effective in reducing cell viability in any cell line, with the only exception of the slight toxic effect on Caco-2 and SW480 lines at the maximum tested concentration. As already mentioned, the antiproliferative activity of cinnamaldehyde on some tumor cell lines was previously described as exerted through different mechanisms, including induction of apoptosis, cell cycle arrest, interruption in angiogenesis, free radical scavenging, inhibition of inflammation, and interference with cellular invasion and metastasis ,26. HoweWhen fraction B antiproliferative activity was tested on E705 cancer cells, by incubating each fraction B at 50 μg/mL concentration in the presence of different cetuximab doses, all fractions showed an additional cytotoxic effect. Interestingly, fraction B from BCHE extract was able to reduce cell viability to almost 50% in the absence of cetuximab and was found to have the same antiproliferative effect at doses of cetuximab between 1 and 10 μg/mL. This finding suggests a potentially very interesting use of cinnamon extracts enriched in polyphenols in chemotherapy, allowing one to reduce cetuximab doses, leading to a decrease in its toxic side effects as well . NotablyTo understand the molecular mechanisms involved in cinnamon polyphenol cytotoxicity towards cancer cells, apoptosis was assessed. All cinnamon fractions B up-regulated the expression levels of cleaved caspase 3 while down-regulating those of Bcl-2. Moreover, they all induced apoptosis increasing ERK phosphorylation in colorectal cancer cell lines.Cinnamomum cassia (BCHE).Contrary to the well-established role of MAPK signaling in promoting cell proliferation and survival, growing evidence suggests that the ERK signaling can mediate proapoptotic signaling. An increasing number of compounds, including betulinic acid, quercetin, kaempferol, and piperlongumine, have been reported to exert apoptosis-inducing effects through ERK activation . Intriguc release, as well as caspase 3 activation [Finally, the decrease in mitochondrial membrane potential, induced by the three cinnamon fractions B is well in accordance with data reported in the literature, showing that cisplatin-induced apoptosis requires ERK activation to induce mitochondrial membrane depolarization and cytochrome tivation . Moreovetivation . Limitat® Isolera™ Prime system . Separations were carried out on the SNAP KP-C18 column using water (A) and methanol (B) as solvents and applying a linear gradient elution (2% B–100% B in 15 CV). Eluate subfractions were pooled in homogeneous groups and solvents were removed under reduced pressure; finally, the residues were dried by lyophilization, obtaining freeze-dried cinnamon polyphenol-enriched fractions.Cinnamon bark and bud extracts and polyphenol-enriched fractions were prepared as previously described by our group . Brieflyhttp://prime.psc.riken.jp/compms/index.html) was used for the peak picking, deconvolution and noise level setting, and identification of metabolites was performed according to their calculated accurate mass and isotopic pattern. Structures were confirmed by comparison MS/MS spectra using the metabolomics MSP spectral kit, public databases, and the literature [High-Resolution Mass Spectrometry (HRMS) analysis of cinnamon polyphenol-enriched fractions (fraction B) was performed using the ACQUITY UPLC H-class system coupled with the Xevo G2-XS QToF Mass Spectrometer  through an ESI source. Samples were dissolved in 90% water–10% acetonitrile at 1 mg/mL and were separated on the ACQUITY Premier HSS T3 Column  coupled with VanGuard™ HSS T3 guard column . The mobile phases were MS-grade water (A) and acetonitrile (B), both containing 0.1% formic acid, and analyte elution was performed according to the following gradient: 0–1 min, 5% B; 1–11 min, 5–50% B linear gradient; 11–12 min, 50–90% B, 12–15 min, isocratic 90% B, and then were equilibrated further for 4 min at the initial conditions (5% B) before the next sample injection. Elution was performed at a flow rate of 0.4 mL/min, and the injection volume was 2 μL. The column temperature was set at 40 °C. Accurate mass data were collected under negative ionization through a data-dependent acquisition mode (FastDDA) in which a full scan survey triggered the MS/MS acquisition of the five most intense ions (Top 5) in the range of 50–1200 m/z. Full scan spectra were acquired at a scan time of 0.2 s and MS/MS spectra were acquired at a scan time of 0.1 s. Dynamic collision energy was set to 6–9 V for 50 Da and 60–80 V for 1200 Da. The source parameters were as follows: electrospray capillary voltage −2 kV, source temperature 120 °C, and desolvation temperature 350 °C. The cone and desolvation gas flows were 50 and 1000 L/h, respectively. The mass spectrometer was calibrated with 0.5 M sodium formate and leucine–enkephalin (100 pg/μL) infused at 10 μL/min and was acquired every 30 s using LockMass. The MassLynx software  was used for instrument control, data acquisition, and data processing. MS Dial software version 4.9.221218  human healthy mucosa cell line and CaCo-2 (ATCC® HTB-37™) human colorectal cancer cell lines were grown in EMEM medium supplemented with heat-inactivated 10% fetal bovine serum (FBS), 2 mM L-glutamine, 0.1 mM non-essential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin. E705  and SW480 (ATCC® CCL-228™) human colorectal cancer cell lines were grown in RPMI 1640 medium supplemented with heat-inactivated 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. All cell lines were maintained at 37 °C in a humidified 5% CO2 incubator. ATCC cell lines were validated by short tandem repeat profiles that were generated by simultaneous amplification of multiple short tandem repeat loci and amelogenin (for gender identification). All the reagents for cell cultures were supplied by EuroClone .CCD841  or Cinnamomum zeylanicum (CZHE) or buds from Cinnamomum cassia (BCHE) at a concentration between 0 and 500 μg/mL and with fractions enriched in polyphenols (B) or cinnamaldehyde (C) at concentrations between 0 and 100 μg/mL. All samples were solubilized in 10% dimethyl sulfoxide (DMSO). The DMSO concentration in the wells was 0.25% for both treated and control cells. Furthermore, 24 h after the seeding, E705 cells were treated with different concentrations of cetuximab , at a fixed concentration of cinnamon fraction B (50 μg/mL). After 48 h at 37 °C, the medium was replaced with a complete medium without phenol red, containing 10 μL of 5 mg/mL MTT -2,5-Diphenyltetrazolium Bromide). After 4 h incubation for CCD841 and 2 h for CRC cell lines, formazan crystals were solubilized with 10% Triton X-100 and 0.1 N HCl in isopropanol, and absorbance was measured at 570 nm using VICTOR® Multilabel Plate Reader . Cell viability was expressed as a percentage against untreated cell lines used as controls. Cell viability assay was investigated using an MTT-based in vitro toxicology assay kit , according to the manufacturer’s protocols. The different cell lines were seeded in 96-well microtiter plates at a density of 1 × 104 cells/well. After 24 h of incubation, the cells were treated for 48 h with cinnamon fraction B at 50 μg/mL obtained from hydroalcoholic extracts of the bark from Cinnamomum cassia (CCHE) or Cinnamomum zeylanicum (CZHE) or the buds from Cinnamomum cassia (BCHE). After treatment, cells were harvested by trypsinization and stained with Annexin V/FITC and propidium iodide (PI) in a binding buffer, according to the manufacturer’s protocol . Cells were analyzed using a flow cytometer, and flow cytometric data were analyzed using CytExpert 2.3 Software .The cells were seeded into 24-well plates at a density of 8 × 106 cells/100 mm dish, and 24 h after seeding, were treated with cinnamon fraction B at 50 μg/mL obtained from hydroalcoholic extracts of the bark from Cinnamomum cassia (CCHE) or Cinnamomum zeylanicum (CZHE) or the buds from Cinnamomum cassia (BCHE) for 48 h. After treatment, the cells were rinsed with ice-cold PBS and lysed in RIPA buffer  containing 1 μM leupeptin, 2 μg/mL aprotinin, 1 μg/mL pepstatin, 1mM PMSF, and a phosphatase inhibitor cocktail . After lysis on ice, homogenates were obtained by passing 5 times through a blunt 20-gauge needle fitted to a syringe and then centrifuged at 15,000× g for 30 min. Supernatants were analyzed for protein content by the BCA protein assay [w/v) dried milk in PBS, probed overnight at 4 °C with the appropriate primary antibodies, and visualized using the ECL detection system . Protein levels were quantified by densitometry of immunoblots using Scion Image software v. 4.0 . The following primary antibodies were used: anti-P-ERK (dilution 1:1000), anti-ERK (dilution 1:1000), anti-BCL2 (dilution 1:1000), anti-caspase 3 (dilution 1:1000) , and anti-vinculin  . IgG HRP anti-rabbit and anti-mouse conjugated secondary antibodies  were diluted 1:5000.For Western blot analysis, the cells were seeded at 1 × 10in assay . SDS-PAGin assay . Sixty m4 cells/well, cultured in complete medium, and, after 24 h, treated with cinnamon fraction B at 50 μg/mL, obtained from hydroalcoholic extracts of bark from Cinnamomum cassia (CCHE) or Cinnamomum zeylanicum (CZHE) or buds from Cinnamomum cassia (BCHE) for 48 h. After treatment, cells were incubated with 40 nM DiOC6 (diluted in PBS) for 20 min at 37 °C in the dark and rinsed with PBS; fluorescence was measured, following PBS addition , using VICTOR® Multilabel plate reader .MTP alterations were assayed through fluorescence analysis, using the green fluorescent membrane dye 3,3′-dihexyloxacarbocyanine iodide (DiOC6), which accumulates in mitochondria due to their negative membrane potential and can be applied to monitor the mitochondrial membrane potential. The different cell lines were seeded in 96-well microtiter plates at a density of 1 × 10p < 0.05.All the experiments were carried out in triplicate. The samples were compared to their reference controls and the data were tested by Dunnett’s multiple comparison procedure (GraphPad Prism Software v. 6.01). Results were considered statistically significant at Collectively, our findings imply that cinnamon polyphenol-enriched fraction has great potential in being utilized for the prevention and treatment of CRC. Cinnamon buds, whose polyphenolic fraction differs significantly from that of the barks, are the matrix with the best biological activity. This is also very interesting since, among the different parts of the cinnamon plant, the buds, to date, are by far the least studied.Moreover, the knowledge of the specific effects of diet components can be useful in the prevention and treatment of several diseases, including gastrointestinal disease and cancer. In this scenario, this work provides the rationale for the use of specific dietary components for prevention and personalized adjuvant therapies.


Background: The inhibition of ubiquitin-specific proteases (USPs) is a novel and promising direction in the development of molecularly targeted therapies in oncology. The aim of the present study was to examine whether Degrasyn could be a potential therapeutic agent against bladder cancer (BC). Also, we aimed to determine whether Degrasyn is more effective in terms of anti-cancer activity compared to the non-selective DUB inhibitor PR-619. To facilitate the translational value of the obtained results, our experiments were performed using both human and canine in vitro models of BC. Methods: Human T24  and SV-HUC-1 , as well as canine K9TCC-PU-NK and RDSVS-TCC1  cell lines, were used in the present study. Cell proliferation was determined using the MTT assay and Ki-67 proliferation assay, and the level of apoptosis induced by Degrasyn and PR-619 was evaluated by Annexin V-FITC staining and caspase 3/7 activation assay. Western blot was used to assess DNA damage and key proteins involved in apoptosis. Results: Degrasyn inhibited the proliferation of all BC cell lines in a concentration- and time-dependent manner. Lower concentrations of Degrasyn were more potent against human and canine BC cell lines compared to PR-619. Degrasyn induced caspase-dependent apoptosis and triggered DNA damage. PR-619 did not show a significant pro-apoptotic effect. Conclusions: Our results demonstrate that Degrasyn significantly impairs the growth of in vitro models of human and canine BC. Selective USP inhibition with Degrasyn seems to be more effective in reducing BC cell proliferation and inducing apoptosis and DNA damage than non-selective USP inhibition with PR-619.  Bladder cancer (BC) is the most common malignancy of the urinary system, with approximately 500,000 individuals diagnosed annually worldwide . BC is aThe majority of NMIBCs can be managed with total curative intent, while the treatment of MIBCs is very challenging and multimodal. Despite the standard treatment , almost 50% of patients with MIBC will eventually develop metastatic disease, which is associated with poor survival rates . The sysSince its discovery almost 40 years ago, the ubiquitin-proteasome system (UPS) has been the subject of increased research interest, as protein ubiquitination and related post-translational modifications regulate diverse aspects of cellular biology. Generally, the process of ubiquitination requires three different enzymes: E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase) . It can Being the largest family among DUBs with over 50 members, USPs have attracted major attention as potential molecular targets in cancer therapy. To date, pharmacological studies have identified several novel DUB inhibitors for potential clinical use, mainly non-selective multiple DUBs/USPs inhibitors . In the BC is not only a significant health issue in humans, but it also occurs in companion animals, including dogs  . With esAs the inhibition of DUBs/USPs functions is a novel and promising direction in the development of molecularly targeted therapies in oncology, the main aim of the present study was to examine whether Degrasyn could be a potential therapeutic agent effective against bladder cancer (BC). Also, we aimed to determine whether Degrasyn is more effective in terms of anti-cancer activity compared to the non-selective DUB inhibitor PR-619. To facilitate the translational value of the obtained results, our experiments were performed using both human and canine in vitro models based on multidisciplinary collaboration.Human T24  and SV-HUC-1  cell lines were used in the present study. They were acquired from the American Type Culture Collection . T24 and SV-HUC-1 cell lines were maintained in McCoy’s 5a Modified Medium and F-12K Medium , respectively. As a representative in vitro model of canine BC, K9TCC-PU-NK and RDSVS-TCC1 cell lines  were also involved in this study. K9TCC-PU-NK and RDSVS-TCC1 cell lines were generous gifts from Deepika Dhawan  and Maciej Parys  School of Veterinary Studies and The Roslin Institute, University of Edinburgh, Scotland), respectively. Both canine BC cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium .2.All culture media used in our study were supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin , and 10% heat-inactivated fetal bovine serum . All cell cultures were performed at 37 °C in a humidified atmosphere containing 5% CO® Caspase3/7 Green Flow Cytometry Assay by ThermoFisher  was used. Anti-USP5 (sc-390943), anti-USP9X/Y (sc-365353), anti-USP14 (sc-515812), anti-Bcl-2 (sc-7382), and anti-β actin (sc-47778) antibodies were purchased from Santa Cruz Biotechnology , whereas the anti-Bcl-xl (#2764) antibody was purchased from Cell Signaling . The anti-γH2A.X (ab26350) antibody was from Abcam , and anti-mouse/HRP and anti-rabbit/HRP antibodies were bought from Dako .Degrasyn (WP1130) and PR-619 were purchased from Selleck Chemicals . FBS, L-glutamine, penicillin and streptomycin solution, 0.25% trypsin EDTA, trypan blue, poly-L-lysine, ribonuclease A, propidium iodide (PI), RIPA buffer, SigmaFAST Protease Inhibitor Cocktail, and TBST buffer were acquired from Sigma-Aldrich . Annexin V-FITC was obtained from Immunostep . The CellEvent5 cells per mL in 96-well plates  and allowed to attach overnight. Subsequently, Degrasyn and PR-619 were added in increasing concentrations . After incubation for 24, 48, and 72 h with either the compounds or in the medium alone, 20 μL of MTT solution (5 mg/mL) was added to each well. After an additional 4 h, 80 μL of the lysis buffer  was added to each well. After dissolving the content, the absorbance was measured at a reference wavelength of 570 nm using a spectrophotometric microplate reader Spark (Tecan). The values were obtained from 3 independent experiments . Based on the results, the concentration that inhibited 50% of the cell proliferation (IC50) was calculated for all incubation intervals.Cell proliferation was determined using the 3--2,5-diphenyltetrazolium bromide (MTT) assay . Cells were plated at a density of 1 × 102, the fresh culture medium was added to inactivate the trypsin, and cells were collected in flow cytometry tubes. Cells were then labeled using the Ki-67 Proliferation Kit BD Pharmingen(BD Biosciences) according to the manufacturer’s instructions and analyzed using a flow cytometer . Unstained control cells were used for gating to determine the percentage of the proliferation of the Ki-67-positive cells in the samples. Percentages of proliferating (Ki-67 positive) cells were used to calculate the means ± SD for each cell line. The presented results were obtained from three independent experiments.For the cell proliferation assay, cells were plated in 100 mm cell culture plates. When the cultures reached 60% confluence, the media were discarded from the wells and replaced with culture media only or media with added compounds  with increasing concentrations for 24 h. The growth medium was then removed from the wells, the cells were rinsed twice with PBS, and 0.25% trypsin-EDTA was added to detach the cells. After 5 min of incubation at 37 °C and 5% COWestern blot was performed to assess the expression of USP5, USP9X, and USP14 in analyzed cell lines. After 24 h of incubation with increasing concentrations of Degrasyn and PR-619, the expression of proteins involved in apoptosis (Bcl-2 and Bcl-xl) and a marker of DNA damage (γH2A.X) was also evaluated. Briefly, cells were rinsed with cold phosphate-buffered saline (PBS), suspended in lysis buffer , and incubated for 20 min on ice. Subsequently, after centrifuging at 10,000 rpm at 4 °C for 12 min, sodium dodecyl sulfate (SDS) sample buffer was added to the suspensions to clear the supernatants. After boiling for 5 min at 95 °C, the samples were subjected to SDS-polyacrylamide gel electrophoresis on 8–15% gel. For USP5, USP9X, USP14, γH2A.X, Bcl-2, and Bcl-xl expression analysis, transfers onto nitrocellulose membranes were performed using a Semidry Transfer Cell or wet transfer method . After transfer, the membranes were blocked with 1% casein in TBS (or 3% BSA for γH2Ax) at room temperature for 1 h. After blocking, the membranes were incubated overnight at 4 °C with primary antibodies: anti-USP5 (dilution 1:500), anti-USP9X/Y (dilution 1:500), anti-USP14 (dilution 1:500), anti-γH2A.X (dilution 1:1000), anti-Bcl-2 (dilution 1:1000), anti-Bcl-xl (dilution 1:1000), and murine monoclonal anti-β-actin antibody (dilution 1:2000). The membranes were then incubated with the secondary antibody for 1 h at room temperature. Membrane visualizations were performed using ChemiDoc Touch Instruments .5/mL in 96-well plates  and incubated for 24 h with increasing concentrations of compounds. Cells were then collected, suspended in a binding buffer, and stained with Annexin V-FITC for 10 min at room temperature. Subsequently, PI was added, and flow cytometric analysis was immediately performed using a flow cytometers . CellQuest 3.lf.  and CytExpert 2.4  software were used for data analysis.To assess the level of apoptosis induced by Degrasyn and PR-619, cells were seeded at a density of 1 × 10Subsequently, to assess caspases 3/7 activation, the cells were incubated with medium alone or with increasing concentrations of compounds  in conditions similar to the previous test. Following cell collection, the cells were twice PBS-washed and stained in accordance with the manufacturer’s instructions. For active caspase 3/7 detection, CellEventCaspase-3/7 Green Detection Reagent Solution was added to the samples. The cells were then suspended 1 more time in the wash buffer after being washed twice. For flow cytometric analysis, a flow cytometers  were used, and CellQuest 3.lf  and CytExpert 2.4  software were used for data analysis. The presented results were obtained from three independent experiments.p < 0.05.Statistical analyses were performed using the GraphPad Prism 9 software . All data were presented as means with standard deviations (SD). Data from the 2 groups were analyzed by a 2-tailed Student’s t-test, and data from multiple groups were analyzed using a 1-way ANOVA followed by Tukey’s posthoc test. Results were considered significant at At the first step of the research, we analyzed USP5, USP9X, and USP14 expression levels in selected cell lines, as they are targets of the inhibitory effect of Degrasyn. As illustrated in 50 values 2.33 μM and 3.07 μM, respectively, p < 0.05). However, the IC50 values did not significantly differ for longer incubation times  (p < 0.05) after incubation only with the highest concentrations of compounds (5 µM Degrasyn and 10 µM PR-619) (p < 0.05) in SV-HUC-1 and canine BC cell lines than in the T24 cell line for all used concentrations of Degrasyn and PR-619 (p < 0.05).The percentage of Ki-67 positive cells (as compared to the control) was significantly decreased after incubation with 2.5–5 µM Degrasyn and 5–10 µM PR-619 in the T24 cell line ( < 0.05) . For oth PR-619) . General50 value for particular cell lines), we detected a significant increase in the number of apoptotic cells (stained only with Annexin V-FITC for early apoptotic cells and double stained with Annexin-FITC and PI for late apoptotic cells) in all cell lines except the K9TCC-PU-NK cell line   after 24 h of incubation with PR-619 compared to Degrasyn in all analyzed cell lines  . In contll lines . The perTo further assess the role of caspases in the apoptosis induced by Degrasyn and PR-619, we evaluated the activation of caspase 3/7. The results are presented in To determine the impact of Degrasyn and PR-619 on USP5 protein expression level, a Western blot was performed after a 24 h incubation with both compounds in the human T24 cell line . As shown in Because γH2AX is a recognized marker of DNA damage, we measured its levels after 24 h of incubation with increasing concentrations of Degrasyn and PR-619. The involvement of key proteins in cell apoptosis after Degrasyn and PR-619 treatment was analyzed using Western blot . No exprGiven that DUBs and USPs target proteins contain a large number of cell homeostasis regulators as well as products of known oncogenes or tumor suppressor genes, DUBs and USPs inhibition has emerged as an appealing and promising research direction for the development of novel cancer therapies . As DegrInitially, we confirmed the expression of USP5, USP9X, and USP14 proteins in selected BC cell lines, which provided the rationale for the utilization of Degrasyn in a BC setting. We observed the overexpression of USP5 protein in a human BC cell line (T24), as compared to normal urothelial cells. Accumulating data suggest that overexpression of USP5 contributes to tumorigenesis in various cancer types through deubiquitinating and stabilizing oncoproteins, such as p53 in melanoma, c-Maf in multiple myeloma, Slug in hepatocellular carcinoma, or histone deacetylase 2 (HDAC2) in ovarian cancer . We evalHaving analyzed the impact of Degrasyn on cell proliferation, we investigated if Degrasyn may cause any particular type of cell death. We demonstrated that Degrasyn might cause apoptosis in BC cells. Our results were consistent with previous reports on multiple myomas . AdditioAccording to several studies, many USPs are involved in DNA damage repair pathways (DDR) ,20. VariWhile there is great interest in DUBs/USPs as a way to provide a high-precision mechanism for the degradation of their substrate proteins, the number of selective compounds is limited to date ,21. One In our study, we also analyzed the anti-cancer activity of Degrasyn and PR-619 on two canine BC cell lines (representing in vitro models of invasive BC in dogs). Generally, canine MIBC shares many similarities with human MIBC, including protein and gene homology, pathophysiological mechanisms of cancer initiation and progression, drug targets, drug resistance, and potential prognostic and diagnostic biomarkers . The mulOur results demonstrate that Degrasyn significantly impairs the growth of in vitro models of human and canine BC. Selective USP inhibition with Degrasyn seems to be more effective in reducing BC cell proliferation and inducing apoptosis and DNA damage than non-selective USP inhibition with PR-619.


The present review explores the critical role of oxime and oxime ether moieties in enhancing the physicochemical and anticancer properties of structurally diverse molecular frameworks. Specific examples are carefully selected to illustrate the distinct contributions of these functional groups to general strategies for molecular design, modulation of biological activities, computational modeling, and structure–activity relationship studies. An extensive literature search was conducted across three databases, including PubMed, Google Scholar, and Scifinder, enabling us to create one of the most comprehensive overviews of how oximes and oxime ethers impact antitumor activities within a wide range of structural frameworks. This search focused on various combinations of keywords or their synonyms, related to the anticancer activity of oximes and oxime ethers, structure–activity relationships, mechanism of action, as well as molecular dynamics and docking studies. Each article was evaluated based on its scientific merit and the depth of the study, resulting in 268 cited references and more than 336 illustrative chemical structures carefully selected to support this analysis. As many previous reviews focus on one subclass of this extensive family of compounds, this report represents one of the rare and fully comprehensive assessments of the anticancer potential of this group of molecules across diverse molecular scaffolds. Cancer is a generic term used to describe a broad spectrum of diseases characterized by the rapid formation of abnormal cells that grow uncontrollably, extending beyond their usual boundaries and invading adjacent parts of the body while destroying normal tissues ,2,3. ThiFortunately, novel approaches to cancer treatment through a surgical targeting of tumors’ microenvironment, such as longitudinal single-cell profiling of chemotherapy ,19 or ta1) reported in 1882 by Meyer and Janny -3-ones, designed and synthesized by Qu et al. , were exectively . 71 effectively arrests the growth of T24 cells in the G1 stage, possibly influencing cell cycle regulatory proteins, particularly cyclin D1, while also causing a significant increase in the activity of p53, p21, and p16 pyrrolocarbazole oximes against recombinant human VEGF-R2 and TIE-2 receptor tyrosine kinases. Compounds 75, 76, and 77 exhibited activity against both TIE-2  and VEGF-R2 , while also demonstrating favorable pharmacokinetic properties in rats quinoxaline derivatives were also synthesized and evaluated for their antiproliferative effects. Among them, compounds 78 , 79 , 80 , 81 , 82 , and 83  demonstrated the ability to inhibit the growth of MDA-MB231, H1299, PC-3, and Huh-7 cancer cell lines, respectively pyrrolizin-8-oximino derivatives synthesized for their antitumor activities by Guo et al. , severalisplatin .112 (IC50 0.042 μM) and 113 (IC50 0.024 μM) were highly selective for VEGFR-2 compared to other VEGFR family members (VEGFR-1 and VEGFR-3). These compounds also exhibited only marginal inhibition effects on other angiogenesis-related kinases, including FGFR, PDGFR, and Tie2, while demonstrating potent antiproliferative activity, as illustrated with 112  and 113 , against HeLa, HCT116, and A375 cancer cells, respectively -benzoquinone oxime tosylate , 5-isopropyl-5-methyl--benzoquinone oxime tosylate , and 2,5-dimethyl--benzoquinone oxime tosylate  showed significant activity against these cell lines, respectively quinolines-based oximes in vitro for their anticancer activity against MCF7, NCI-H460, and SF-268 cell lines, Chen et al.  revealed process . The pha (3.4 h) . Followier cells .Z)-6-[2-(4-methoxyphenyl)-2-hydroxyiminoethoxy]quinolin-2(1H)-one (188) appeared to be the most potent against AA-induced platelet aggregation, with an IC50 of 0.58 μM, but exhibited no cell proliferation inhibition effects -4-piperidone oximes, prepared as potential antineoplastic agents, were also evaluated against a panel of leukemic human colon cancer cell lines, including Colo205, HCC-2998, HCT-116, HCT-15, HT29, KM12, and SW-620. Among them, compounds ectively . As a ge22 cells .215 (IC50 1.29 and 1.61 μM) and 216 (IC50 10.1 and 14.13 μM), which showed better cytotoxicity than the parent natural product against MDA-MB231 and U2OS cancer cells, respectively, with compound 216 exhibiting an about 130-fold increase in PBS solubility compared to the parent compound benzoyloxy-imino-androst-5-ene (288) emerged as the most potent conjugate [50 of 0.937 µM. Generally, the androstene oxime-nitrogen mustard conjugates derived from the D-ring of the steroid skeleton displayed superior antineoplastic activity compared to those derived from other rings [While evaluating a series of androstene oxime-nitrogen mustard bioconjugates for their cytotoxicity against a panel of 60 cancer cell lines, 17onjugate . It exhier rings .O-phenylacetamide)oxime derivatives for their antiproliferative activity against human hepatocellular carcinoma cells  and human breast cancer cells . Compounds 289 , 290 , and 291  exhibited moderate in vitro antiproliferative activities. Additionally, fluorescence imaging revealed that the coumarin-289 conjugate (292) primarily accumulates in the mitochondria, resulting in a stronger anticancer activity compared to the parent structure [Bu et al.  evaluatetructure .293) encompassing a photosensitizing drug (red-emitting pheophorbide a) and nandrolone, a steroid that specifically binds to the androgen receptor, often overexpressed in various tumors. Since light-triggered therapies could potentially have a significantly positive impact on the treatment of hormone-sensitive cancers, this targeted bimodal oxime conjugate was evaluated on prostatic cancer cell lines in vitro, using an AR-positive cell line (LNCaP) and an AR-negative/positive cell line (PC-3) [293 localizes in the endoplasmic reticulum and mitochondria [293) from the cell interior, providing additional evidence for its binding specificity. Photodynamic therapy is progressively emerging as an alternative treatment for both malignant and non-malignant cancers, despite its limitations in targeting specificity, which is critical for preventing systemic toxicity. Pavlíčková et al.  reportede (PC-3) . U-2 OS chondria . A compeThe clinical success of the anticancer cisplatin, discovered in the 1960s, stimulated a significant interest in the use of metal complexes in cancer treatment ,248. As trans-configured oxime-liganded platinum complexes, which displayed many attributes different from those of established anticancer agents, including a distinct DNA binding behavior, increased cellular accumulation, and an unusual pattern of protein interaction, have gained traction as a new class of anticancer therapies [Furthermore, herapies ,249,251.herapies ,255,256,294 (IC50 3.9 μM), 295 (IC50 16 μM), 296 (IC50 1.0 μM) were primarily active against CH1 cell line, while 297 (IC50 8.3 and 12 μM) showed potency against both CH1 and SW480, respectively [298 was the most potent of the series across the board, exhibiting IC50 values of 1.1, 0.97, and 5.6 μM against CH1, SW480 and A549, respectively [Some of these kinetically inert platinum(II) complexes bearing a chelating oxime ligand were evaluated for their cytotoxic properties on three human tumor cell lines originating from ovarian carcinoma (CH1), colon carcinoma (SW480), and non-small cell lung cancer (A549) . This evectively . Compounectively .cis- and trans-configured acetone oxime platinum(II) complexes, as well as one pair of pentan-3-one oxime platinum(II) complexes. They found that, overall, trans-platinum(II) oxime complexes, 299(t) (IC50 0.22 µM), 300(t) (IC50 2.5 µM), 301(t) (IC50 2.8 µM), and 302(t) (IC50 1.9 µM), exhibited higher cytotoxicity compared to their corresponding cis congeners: 299(c) (IC50 3.4 µM), 300(c) (IC50 45 µM), 301(c) (IC50 1.8 µM), and 302(c) (IC50 15 µM), on the cisplatin-resistant SW480 cell line [trans-configured complexes within SW480 cells was up to 100 times higher than that of cisplatin and up to 50 times higher than their cis-configured counterparts. Furthermore, the ‘r’ values (number of platinum atoms per nucleotide) were more than ten-fold higher in cells treated with trans complexes compared to those treated with cisplatin [Bartel et al.  reportedell line . This stisplatin .cis complexes did not induce any DNA degradation, the trans complexes resulted in pronounced strand cleavage [trans complexes, which potentially contributed to their cytotoxic effect [The interaction of these complexes with DNA was further investigated in cell-free experiments using plasmid DNA (pUC19), in capillary zone electrophoresis with the DNA model 2-deoxyguanosine 5′-monophosphate, and through in vitro experiments measuring the extent of DNA damage in the comet assay. While incubation with cleavage . The mosc effect .trans platinum(II) oxime complexes and their head-to-tail-oriented oximato-bridged dimers featuring a six-membered diplatinacycle and representing a unique class of oxime ligands coordinated to platinum via an oxygen atom, was also evaluated on three human cancer cell lines, including the cisplatin-sensitive CH1 cells as well as cisplatin-resistant SW480 and A549 cells [303 , 304 , 305 , 306 , 307 , 308 , 309 , 310 , and 311 , exhibited strong potency against all the three cell lines [50 values of Pt(II) compounds, with the reverse trend observed for analogous complexes featuring ketoxime ligands. Notably, the dimers did not produce any cross-resistance with cisplatin in SW480 cells, as it demonstrated an up to two-fold increase in cytotoxicity in comparison with its effect on the CH1 cell line [305 as well as one dimer 309 proved to be strong apoptotic agents capable of inducing cell death even in the cisplatin-resistant SW480 cell line [305, 306, 309, and 310) instead of acetaldehyde oxime  did not result in a significant increase in cytotoxicity. However, the E- and Z- configuration of the coordinated aldoximes appeared to have some impact on the obtained IC50 values, as monomeric complexes 304 and 306 featuring both E-and Z-aldoximes exhibited an up to two-fold increase in cytotoxicity against CH1 cells compared to their isomers 303 and 305, containing two Z ligands [The cytotoxicity of some 49 cells . All thell lines . The dimell line . Furtherell line . Increas ligands .312 with IC50 values of 3.87, 5.18, 3.83, 14.1, 0.62, 1.43, and 3.15 μM, and 313 with IC50 values of 2.47, 2.50, 2.63, 3.61, 0.31, 1.08, and 2.60 μM, exhibited potency against a panel of human tumor and mesothelial cells, including HeLa, A549, HT-29, MSTO-211H, A2780, A2780cis, and Met-5A, respectively [313 exhibited an outstanding cytotoxicity in ovarian carcinoma cells (A2780), with a significant and unexpected antiproliferative effect on cisplatin-resistant cells (A2780cis). Further studies into the intracellular mechanism of action indicated that 313 had a lower ability to bind to DNA compared to cisplatin (used as a control), but demonstrated a noticeably higher uptake in resistant cells [313 [Pt(II) complexes bearing triphenylphosphine and chelating oximes were found to efficiently penetrate resistant cells and induce significant antiproliferative effects, despite minimal interaction with DNA . Among tectively . Notablynt cells . A highells [313 .314  cells, with 319 showing statistically significant in vivo activity in DL-bearing mice.Half-sandwich ruthenium, rhodium, and iridium complexes synthesized from aldoxime, ketoxime, and amidoxime ligands also exhibited in vitro antitumor activity against HT-29, BE, and MIA PaCa-2 cell lines, especially for complexes 325 (IC50 24.4 μM), 326 (IC50 14.8 μM), and 327 (IC50 21.5 μM) showed considerably higher cytotoxicity than cisplatin (IC50 510 μM) under the same experimental conditions [While investigating the ability of water-soluble chiral arene Ru(II) complexes bearing amino-oxime ligands to interact with plasmid (pBR322) DNA and to exhibit cytotoxic and antimetastatic effects in vitro against androgen-unresponsive human prostate cancer PC3 cell lines, Benabdelouahab et al.  discovernditions .E)-3-hydrazono)butan-2- one oxime, respectively. These compounds included iron (328), silver (329), zirconium (330), uranium (331), cobalt (332), and copper  complexes, all of which were evaluated for their anticancer effects. Among them, copper complexes, including 334 (IC50 6.49 μM), 335 (IC50 2.67 μM), and 336 (IC50 2.24 μM), as well as the cobalt complex 332 (IC50 36.8 μM), exhibited noticeable antiproliferative potency against HepG2 human liver cancer cell lines [Furthermore, Saad El-Tabl et al. ,267 and ll lines ,267.This report represents one of the most comprehensive assessments of the anticancer potential of structurally diverse molecular frameworks bearing oxime and oxime ether moieties. Specific examples have been carefully selected to illustrate the distinct contributions of these functional groups to general strategies for molecular design, modulation of biological activities, computational modeling, and structure–activity relationship studies. The primary focus is on the role of this functional group in enhancing the physicochemical and anticancer properties of the resulting molecules. Additional mechanistic investigations, molecular dynamics, and docking studies indicate that the mechanism and the potential drug targets of the resulting compounds also depend on the chemical framework to which the functional group is attached. In some cases, the oxime or oxime ether groups enhanced the already available anticancer properties, while in others, these groups were fully responsible for the observed properties, as without this functional group this scaffold was completely inactive. The pharmacokinetic and pharmacodynamic profiles, as well as drug-likeness, also appeared to be impacted to a greater extent by the addition of the oxime or oxime ether functional group. Regardless of the situation, these compounds appear to inhibit, interact with, or interfere with a number of key markers in cancer pathogenesis and malignancy. As a result, it is not unlikely that some of these oxime and/or oxime ether-bearing molecular frameworks could soon advance to clinical trials or even make it to the market.