The newly synthesized enoxacin derivatives have shown a magnified cytotoxic effect with an emphasis on prooxidative, proapoptotic and microRNA interference actions. The mentioned mechanisms seem to contribute to a safer, more selective and more effective anticancer therapy. Enoxacin is a second-generation quinolone with promising anticancer activity. In contrast to other members of the quinolone group, it exhibits an extraordinary cytotoxic mechanism of action. Enoxacin enhances RNA interference and promotes microRNA processing, as well as the production of free radicals. Interestingly, apart from its proapoptotic, cell cycle arresting and cytostatic effects, enoxacin manifests a limitation of cancer invasiveness. The underlying mechanisms are the competitive inhibition of vacuolar H+-ATPase subunits and the c-Jun N-terminal kinase signaling pathway, causing a decrease in cell invasiveness. Interestingly, the prooxidative nature of the subjected fluoroquinolone enhanced the cytotoxic effect. Crucial for the anticancer activity were the carboxyl group at the third carbon atom, fluorine at the seventh carbon atom and nitrogen at the eighth position of naphyridine. Modifications of the parent drug improved the induction of oxidative stress, cell cycle arrest and the dysregulation of microRNA. The inhibition of V-ATPase–microfilament binding was also observed. Enoxacin strongly affected various cancer but not normal cells, excluding keratinocytes, which suffered from phototoxicity. It seems to be an underestimated anticancer drug with pleiotropic action. Furthermore, its usage as a safe antibiotic with well-known pharmacokinetics and selectivity will enhance the development of anticancer treatment strategies. This review covers articles published within the years 2000–2021, with a strong focus on the recent years (2016–2021). However, some canonical papers published in twentieth century are also mentioned.Enoxacin as a second-generation synthetic quinolone is known for its antibacterial action; however, in recent years there have been studies focusing on its anticancer potential. Interestingly, it turns out that compared to other fluoroquinolones, enoxacin exhibits uncommon cytotoxic properties. Besides its influence on apoptosis, the cell cycle and cell growth, it exhibits a regulatory action on microRNA biogenesis. It was revealed that the molecular targets of the enoxacin-mediated inhibition of osteoclastogenesis are vacuolar H Fluoroquinolones are synthetic antibacterial agents, representing the first set of anti-infective agents that were not modeled knowingly after any natural antibiotics ,2. Over Enoxacin (ENX)  is an orFluoroquinolones are a group of substances that have been thoroughly tested in terms of their anticancer activity. Only in recent years have a number of reviews describing the cytotoxic properties of fluoroquinolones been published ,12,13,14Like most fluoroquinolones, enoxacin has been tested for its anticancer activity. However, distinct activities of enoxacin, such as anti-ALS, antidepressant, antiaging and antiviral activities, are reported in literature . In thisThe miRNA biogenesis begins with transcribing a gene into a large primary transcript (pri-miRNA). The transcription is typically mediated by RNA polymerase II . The priIt has been clear for almost twenty years that miRNA expression is dysregulated in human malignancies. The underlying mechanisms include chromosomal abnormalities ,26,27,28Small molecules can influence miRNA biogenesis. Enoxacin was reported as the first and unique small-molecule enhancer of microRNA (SMER) maturation . Jin et al. found that the increase in miRNAs was associated with high levels of their own precursors, thereby suggesting that enoxacin could promote the DICER processing activity without influencing the miRNA precursor expression . The samThe role of the TRBP in enoxacin-mediated cytotoxicity was confirmed in the three colorectal cancer cell lines  and their mutants with impaired TRBP expression. TRBP-impaired cells were more resistant to enoxacin, resulting in a 2-fold increase in the effective concentration (EC50). Mutant cells also did not undergo cell cycle arrest. Additionally, enoxacin induced a two-fold expression increase in 24 miRNAs in RKO and HCT-116 cells and from 1.5-fold up two five-fold increases in the expression of those miRNAs in RKO and HCT-116 xenograft mouse model. Enoxacin treatment also significantly decreased the number of lung and liver metastases in the HCT-116 xenograft . InteresThe role of enoxacin as an enhancer of DNA damage response (DDR) signaling was confirmed in cervical cancer HeLa cells. This antibiotic augmented the DDR mediator factors pATMS1981, 53BP1, MDC1 and pS/TQ without affecting their expression, whereas the activity of γH2AX levels was not affected. Based on the knowledge of the activation of TRBP by enoxacin, it is not surprising that TRBP silencing diminished ENX’s stimulation of DDR. The knockdown of PACT or GW182 proteins , which are effectors for miRNA-guided gene silencing, did not affect ENX-mediated DDR stimulation. These results seem to strengthen and confirm enoxacin’s specificity towards TRBP .Regarding the molecular target recognized by enoxacin, an additional protein has been proposed that merits mentioning. In 2017, Abell et al. determined that the Piwi-like protein 3 (PIWIL3) is a potential enoxacin target . PIWIL3 Additionally, ENX affected RNA helicase DHX9, a member of the RISC. The expression of DHX9 was much higher in the small-cell lung cancer cell line H446 than in the non-small-cell lung carcinoma cell lines A549 and PC9. Enoxacin inhibited the proliferation of A549 in a dose-dependent manner. Similarly, it decreased the expression of DHX9 in A549 cells. However, silencing DHX9 impaired the cytotoxic effect of the drug . A strong inhibitory effect of enoxacin on the proliferation of human melanoma A375, Mel-Juso and Mel-Ho cell lines was observed. It dysregulated a set of 55 miRNAs in A375 cells . Two upregulated miRNAs, miRNA-3154 and miRNA-4459, control the p53-Mdm2-MdmX network . InteresA different ENX antiproliferative mechanism without affecting apoptosis has been noted in 4T1 murine breast cancer cells. An increased level of GW/processing bodies, which are considered the surrogate markers for both the microRNA-mediated repression of translation and the extracellular vesicle (EV) packaging sites, was observed. MiRNA expression levels in the 4T1 cells’ cytosol and their EV were compared. Enoxacin significantly increased only miRNA-214-3p and slightly increased miRNA-146a-5p, miRNA-290, miRNA-689 and let-7b-5p cytosolic levels. In contrast, significant decreases in let-7b-5p, miRNA-146a-5p and miRNA-689 expression and an enormous 22-fold increase in miRNA-214-3p were observed. All mentioned miRNAs are involved in the regulation of bone remodeling and osteoclastogenesis. Additionally, EVs from enoxacin-treated 4T1 cells enhanced the proliferation of murine macrophage cells . The effAlthough it has been clear that enoxacin did not affect all existing miRNAs  CD8 cells from an autoimmune cholangitis mouse model. Despite the fact that enoxacin did not change the amount of CD8 T cells, it significantly decreased their proliferative response. Enoxacin also significantly decreased the level of interferon γ in mouse serum . There iEnoxacin is also capable of affecting artificial miRNAs (amiRNAs), especially by enhancing the amiRNA-mediated reversible inhibition of the CRISPR-Cas9 system in both in vitro and in vivo studies. Interestingly, amiRNA alone did not show an inhibitory effect towards the CRISPR-Cas9 system, indicating the crucial role of enoxacin. In contrast, some of the naturally occurring miRNAs were able to inhibit CRISPR-Cas9 activity by binding to single-guide RNA (sgRNA), a part of the sgRNA/Cas9 complex, in the absence of enoxacin. Surprisingly, the presence of enoxacin in concentrations up to 50 µM did not affect the natural miRNAs’ influence on the CRISPR system. The proposed explanation of these differences in the impacts of enoxacin on the inhibition of natural and artificial miRNAs is based on the low binding capacity of amiRNAs towards RISC and the high binding capacity towards the RISC of natural miRNAs. Another factor could be the difference in the amounts of amiRNAs vs. natural miRNAs. The amiRNAs are believed to outnumber natural miRNAs. Taking the above into account, the amiRNA/enoxacin system turned out to be specific and reversible, making it a convenient tool for CRISPR-Cas9 regulation .In 2008, Shan et al. investigated the ability of microRNA processing to enhance some fluoroquinolones, including enoxacin and its three derivatives. They identified the structural elements of enoxacin responsible for its activity, such as a carboxyl group at the 3rd carbon atom, fluorine at the 7th carbon atom, as well as nitrogen at the 8th position of naphyridine. .Enoxacin affected cancer cells via various mechanisms . It indu+-ATPase (V-ATPase) and microfilaments. The affected subunits of V-ATPase were B2 subunit ..69].LZ-106 induced apoptosis via activation of the ROS-dependent DNA damage response in NSCLC  in both cultured cells and in a xenograft mouse model. The authors demonstrated that LZ-106 notably induced ROS overproduction and DDR. Interestingly, additional evidence in their findings revealed that DDR and apoptosis could be alleviated in the presence of an ROS scavenger, N-acetyl-cysteine (NAC), indicating ROS-dependent DDR involvement in the LZ-106-induced apoptosis. This research not only offered a new therapeutic candidate for NSCLC, but also gave new insights into the pharmacological research of quinolones .In 2019, Yang et al. continued to investigate the mechanism of the cytotoxic activity of LZ-106. They substantiated the involvement of P53 activation in intracellular ROS generation upon LZ-106 treatment and related P53 to the ROS-induced viability inhibition and apoptosis, which was presented in previous research. P53 was shown to play an indispensable role in the upregulation of intracellular ROS in LZ-106-treated NSCLC cells through ROS detection. The authors further identified the antiproliferation effect of LZ-106 in NSCLC cells exhibited through G1 phase cell cycle arrest with a cell cycle analysis, with an expression analysis of the key proteins, and discovered that the cell cycle arrest effect is also mediated by the induction of ROS in a P53-dependent manner. In addition, the tumor-suppressive effect exhibited in vivo was demonstrated to be similar to that in vitro, which requires the participation of P53. Thus, LZ-106 is a potent antitumor drug possessing potent proliferation inhibition and apoptosis induction abilities through P53-dependent ROS modulation both in vitro and in vivo .In 2018, Vracar et al. showed that enoxacin and bis-enoxacin  stimulatBis-enoxacin (BE) did not induce apoptosis or inhibit 4T1 murine breast cancer cell proliferation; however, it altered the release of extracellular vesicles and altered the miRNA expression. It also increased the number of GW/P bodies. Both enoxacin and bis-enoxacin at a concentration of 50 µM increased the number of GW/P bodies, but there were minimal changes in microRNA levels. Extracellular vesicles (EVs) released from 4T1 cells treated with 50 µM enoxacin or 50 µM bis-enoxacin stimulated the proliferation of RAW 264.7 cells, and both drugs significantly inhibited osteoclastogenesis in calcitriol-stimulated mouse marrow. EVs from 4T1 cells treated with enoxacin and bis-enoxacin displayed small reductions in the amounts of miRNA-146a-5p and let-7b-5p. In marked contrast, miRNA-214-3p, which has been shown to regulate bone remodeling, was increased 22-fold and 30-fold, respectively. We conclude that enoxacin and bis-enoxacin trigger in 4T1 cancer cells the release of EVs that inhibit osteoclastogenesis . SimilarThe introduction of 2-(5-chlorothiophen-2-yl)ethyl into the piperazine ring of enoxacin  increase50 range for NOH of ca. 3–10 µM and a IC50 range for NOMe of ca. 3–20 µM (depending on the cell line). A similar IC50 range of 2–14 µM for NOBn derivatives was observed in melanoma, epidermoid, cervical and bladder carcinoma cells. The IC50 values of NOBn derivatives in other cell lines and those of oxygen in all tested cell lines were much higher, but still significantly lower than the IC50 of unmodified enoxacin. To sum up, the introduction of the 2-(5-chlorothio-phen-2-yl)ethyl moiety into the 4′ position of enoxacin increased the cytotoxicity of the compound; however, the strength of the effect depended on the structure of the ethyl spacer. Introducing another nitrogen atom  led to a significant increase in antitumor activity [The synthetized derivatives differ from each other in the modification of the structure of an ethyl spacer. The introduction of oxygen into the ethyl spacer increased the cytotoxic activity from 137–196 µM for enoxacin to 137–131 µM for the oxygen derivative, which was a relatively minor change. The introduction of NOH or NOMe led to the strongest cytotoxic activity vs. all tested cell lines, with a ICactivity . A series of enoxacin derivatives was formulated via the transformation of the carboxyl group in the skeleton of N-methylenoxacin (position 3) to an aldehyde group, followed by a direct reaction with N-arylhydrazinecarbothioamide (thiosemicarbazide) (CN106674220) or subjected to a condensation reaction with methyl hydrazinodithioformate and next to a nucleophilic substitution reaction with some N-alkylamines ,105. TheThe obtained derivatives were tested for antitumor activity on cancer cell lines, including Hep-3B (human liver cell line containing integrated hepatis B virus genome), PANC-1 (human pancreatic cancer cell line) and HL-60 (human leukemia cell line). The cytotoxic activity against cancer cell lines was compared to that against a normal cell line—Vero . 50 6.0–20.5 µM). The most active derivative was the derivative with the cyclopropyl substituent in the tiosemicarbazide part. Extending the alkyl substituent, especially above four carbon atoms, resulted in lower cytotoxic activity. All derivatives were characterized by significantly lower cytotoxicity against the normal Vero cell line (over 66.5 µM) [The alkyl derivatives showed cytotoxicity within limits (IC66.5 µM) .50 3.2–6.2 µM). In this case, all derivatives were also characterized by significantly lower cytotoxicity compared to the normal Vero cell line (above 73.4 µM) [The aryl derivatives, similar to the alkyl ones, were characterized by much higher cytotoxicity compared to enoxacin, in the range of 2.7 to 33.5 µM. Derivatives 1a–5a and 7a are characterized by more than two times lower cytotoxic activity compared to derivatives 6a and 8a–11a. The introduction of fluoro substituents to the phenyl ring or the replacement of the phenyl substituent with pyridyl, furanyl and thiophenyl substituents increases the cytotoxic activity. The most active compound was the derivative containing a pyridyl substituent in the tiosemicarbazide part (IC73.4 µM) .50 1.8–21.5 µM. The derivatives numbered 1–4 are characterized by more than two times lower cytotoxic activity compared to the derivatives numbered 5–10. The most active derivative was the derivative containing a benzenesulfonamide substituent in the rhodanine part (10). In this case, all derivatives were also characterized by a significantly lower cytotoxicity compared to the normal Vero cell line (above 58.3 µM) [Chinese scientists have tested the cytotoxic activity of some rhodanine derivatives of N-methylenoxacin . The met58.3 µM) .Another patented derivative was a dimeric form of enoxacin. The 1-(N-enoxacin amide)-enoxacin derivative  was obtaEnoxacin is a multifaceted drug. It is not only an effective antibiotic but also a promising anticancer agent. Unlike other fluoroquinolones, ENX exhibits extraordinary cytotoxic mechanisms of action. The presence of a carboxyl group at the third carbon atom, fluorine at the seventh carbon atom, as well as nitrogen at the eighth position of naphyridine is crucial for the regulation of miRNA abundance. ENX has the potential for the dysregulation of miRNA biogenesis by interfering with the PIWIL3 protein or DHX9 helicase, a part of the RNA-induced silencing complex (RISC), as well as the RISC-loading complex proteins DICER and TRBP. Additionally, it induced an elevated number of GW/P bodies. The alteration of those pathways was confirmed in the enoxacin-induced cytotoxicity towards colorectal, breast, prostate, thyroid and Ewing’s sarcoma family (ESFT) cancer cells. Changes in miRNA biogenesis resulted also in a decrease in inflammatory response in mice, affecting the neurobiology, including in neuronal differentiation and the inhibition of osteoclastogenesis. Another confirmed mechanism of osteoclast bone resorption inhibition was the suppression of the JNK signaling pathway and binding with V-ATPase subunits. Moreover, it has been described as a convenient tool for the regulation of CRISPR-Cas9 activity. Another anticancer mechanism was the induction of DNA damage by ROS stress generation. Enoxacin caused an induction of oxidative stress by singlet oxygen but not by superoxide radicals. Interestingly, it induced morphological changes, cell cycle arrest, growth inhibition, a decrease in invasiveness and apoptosis in cancer but not in normal cells. The above findings give evidence for the use of enoxacin, especially in bladder cancer therapy, where the achievable urine concentrations are far higher than IC50 and can be maintained constantly for a long time period. Furthermore, this type of cancer could be harnessed using two sources of enoxacin—urine and blood.The modification of the piperazine ring or the carboxylic group resulted in enhanced cytotoxicity comparing to free enoxacin. Some of the derivatives were proven to cause cell cycle arrest and the induction of oxidative stress, while others dysregulated the levels of miRNA. Interestingly, the LZ-106 derivative caused the formation of superoxide radicals, in contrast to the parent drug. Unfortunately there are no clinical studies on enoxacin’s anticancer activity, while the results from in vitro studies are scarce compared to other fluoroquinolones. Taken together, enoxacin has anticancer potential, and its modifications may bring additional therapeutic benefits.


Pinus koraiensis), using cell lines with both high (F: 001#-001) and low (S: 001#-010) proliferation potential. We found that exogenous GSH promoted cell proliferation in both cell lines, while exogenous BSO inhibited proliferation in both cell lines. At 35 d with exogenous GSH treatment, the fresh weight of F and S cell lines increased by 35.48% and 48.39%, respectively, compared with the control. The exogenous application of GSH increased the intracellular levels of GSH, total GSH (T-GSH), oxidized glutathione (GSSG), ascorbic acid (ASA), total ASA (T-ASA), and the ratios of GSH:T-GSH and ASA:T-ASA in both F and S cell lines. Furthermore, exogenous GSH increased the activity of both glutathione reductase (GR) and dehydroascorbate reductase (DHAR) while decreasing the activity of ascorbate peroxidase (APX) in both cell lines. It appears that the application of exogenous GSH promotes a reducing cultural environment, which is conducive to EC proliferation in Korean pine. By helping to reveal the mechanism whereby GSH regulates redox homeostasis in Korean pine EC cells, we have laid the foundation for a large-scale breeding of Korean pine somatic embryogenesis technology system.Somatic embryogenesis (SE), which leads to the formation of embryonic callus (EC) tissue, is the most promising method for large-scale production and selective breeding of woody plants. However, in many species, SE suffers from low proliferation rates, hindering the production of improved plant materials. One way of improving proliferation rates is achieved by improving the redox status of the culture medium. In this study, we investigated the effects of exogenous glutathione (GSH) and L-buthionine sulfoximine  on the EC proliferation rate in Korean pine ( Somatic embryogenesis (SE) is the artificial formation of embryo-like structures from somatic cells without the need for gametic fusion . SE is oPinus koraiensis Sieb. et Zucc.) is a dominant species in mixed-broadleaved Korean pine forests in Northeast China [Korean pine  [Picea glauca), regulation of the redox status of the SE medium by application of exogenous GSH or GSSG promoted a dramatic increase in both EC proliferation (25% by weight) and the number of early filamentous embryos, accompanied by an increase in ATP and nucleotide production. Inhibiting GSH synthesis through the application of L-buthionine sulfoximine (BSO) has been shown to induce a pro-oxidative environment, thereby reducing the cellular GSH: total glutathione (T-GSH) ratio [Cellular antioxidants such as glutathione (GSH)/oxidized glutathione (GSSG) and ascorbic acid (ASA)/dehydroascorbic acid (DHA) play an important role in many biological processes, including cell division and differentiation, and can impact the process of SE through changes in cellular reduction oxidation (redox) status ,19,20,21ylifera) . In whitH) ratio . Both ASThe proliferative capacity of EC in conifers is essential for the large-scale production of high-quality seedlings. GSH promotes EC proliferation in white spruce, but it is unclear whether GSH has an effect on EC proliferation in Korean pine. The effect of exogenous GSH on the accumulation of GSH and ASA in EC cells of Korean pine and the cycling between them is not clear. In this study, we investigated the effects of exogenous GSH and BSO on the EC proliferation rate in Korean pine, using cell lines with both high (F: 001#-001) and low (S: 001#-010) proliferation potential. Furthermore, we examined redox regulation, as well as intracellular GSH and ASA synthesis, in these cell lines. This work will serve to optimize large-scale breeding efforts in woody plants by improving SE efficiency, particularly in Korean pine and other conifer species.p < 0.05) (The growth status of the two cell lines is shown in  < 0.05) . Exogeno < 0.05) a, wherea < 0.05) b. The FW < 0.05) a. At 35 p < 0.05) (The effects of exogenous application of GSH and BSO on EC dry weight (DW) of the two cells lines were different ( < 0.05) . Overall < 0.05) a, while  < 0.05) b. The DW < 0.05) a. The DWp > 0.05)  .The exogenous application of GSH promoted T-GSH synthesis in both cell lines a,b. In tp < 0.05) (The effects of exogenous application of GSH and BSO on the intracellular GSH content of the two cell lines were different ( < 0.05) c,d. The  < 0.05) c. The ex < 0.05) d. The inp < 0.05) (The effects of exogenous application of GSH and BSO on the intracellular GSSG content of the two cell lines were different ( < 0.05) e,f. Exog < 0.05) e. The ex < 0.05) f. The inThe exogenous application of GSH increased the intracellular GSH:T-GSH ratio in F and S cell lines g,h, withThe exogenous application of GSH promoted the synthesis of intracellular T-ASA in F and S cell lines a, with ip < 0.05) (The effects of exogenous application of GSH and BSO on the intracellular ASA content of the two cell lines were different ( < 0.05) c,d. The  < 0.05) c. The in < 0.05) d, with tp < 0.05) (The effects of exogenous application of GSH and BSO on the intracellular DHA content of the two cell lines were differed ( < 0.05) e,f. The  < 0.05) e, with t < 0.05) f, with tThe exogenous application of GSH tended to increase the intracellular ASA:T-ASA ratio in F cell lines g, althoup < 0.05) (The effects of exogenous application of GSH and BSO on intracellular GR activity of the two cell lines were differed ( < 0.05) a,b. Intr < 0.05) a. Intrac < 0.05) a. The ex < 0.05) b. Intracp < 0.05) (The effects of exogenous application of GSH and BSO on intracellular APX activity of the two cell lines were differed ( < 0.05) c,d. Intr < 0.05) c. At day < 0.05) d, with ip < 0.05) (The effects of exogenous application of GSH and BSO on intracellular DHAR activity of the two cell lines were differed ( < 0.05) e,f. In t < 0.05) e. Simila < 0.05) f. IntracTen differentially expressed Korean pine genes were selected for qRT-PCR validation . Of thes. Healthy EC tissue and a fast proliferation rate are essential during SE. To that end, GSH has been shown to regulate cell division and differentiation in many organisms, and has been found in high concentrations in rapidly proliferating cells [P. glauca) SE have shown that exogenous application of GSH promotes the proliferation of EC, increasing EC FW by as much as 25%. In this work, we examined the effect of exogenous application of GSH and BSO on Korean pine SE and found that GSH promoted EC proliferation, while BSO inhibited EC proliferation, compared to CK.EC material allows for efficient genetic transformation, makes an ideal system for studying single-cell differentiation and allotropic expression, and can form the basis for large-scale production of woody plants . Healthyng cells . More spng cells . StudiesGSH is present in most prokaryotic and eukaryotic cells  and playNicotiana tabacum), exogenous application of ASA during cell division has been shown to increase intracellular soluble protein content, and decrease both malondialdehyde (MDA) content and cell membrane permeability [ASA is the primary component of the ASA-GSH cycle and plays an important role in cellular growth, elongation, and division frequency, and also in protecting the cell membrane . In tobaeability . ASA is GSH is an electron receptor for DHA and plays an important role in scavenging intracellular ROS and maintaining redox homeostasis in plants . In bothEupatorium adenophora) have been found to upregulate GR activity when exposed to low temperatures, but not high temperatures [Eupatorium odoratum) show the opposite trend. In white spruce (P. glauca), the exogenous application of BSO during SE increased intracellular GR activity [Brassica napus), the exogenous application of BSO during SE reduced intracellular GR activity [GR is a flavinase which catalyzes the formation of GSH from GSSG. GR also plays a crucial role in maintaining cellular redox homeostasis by scavenging intracellular ROS. Previous studies have found that GR activity increases when plants are subjected to stressful environmental conditions, including exposure to ozone, drought, heavy metals, bright light, salt, and low temperatures. Additionally, the GR activity of stress-tolerant plant species remains high during stress exposure, but drops in susceptible species . Plants eratures . Contraractivity , while iactivity . In thisCasuarina equisetifolia) [Pisum sativum) [Citrus reticulata) [Oryza sativa) [Solanum tuberosum) varieties, while APX activity decreased in salt-sensitive varieties. In oilseed rape (B. napus), APX activity was higher in embryos treated with exogenous BSO during the globular embryo period, and APX activity could be modified by exogenous GSH [In the ASA-GSH cycle, ASA is converted by APX to monodehydroascorbic acid (MDHA) . Changestifolia) , pea (Pisativum) , citrus iculata) , and ric sativa) . Additionous GSH . These rnous GSH . In thisDHAR is a key enzyme in the ASA-GSH cycle, which promotes ASA regeneration and serves to protect cells against oxidative damage. Specifically, DHAR catalyzes the production of ASA from GSH, thus helping to regulate the ratio of intracellular ASA to DHA . In thisTwo Korean pine cell lines, one with high proliferative potential 001#-001 (F) and one with low proliferative potential 001#-010 (S), were revived and cultured after ultra-low temperature preservation. EC induction was performed using mLV ,49 mediuAll culture media in this study had a pH of 5.8 and were autoclaved  after pH adjustment. After the medium was sterilized and cooled to approximately 55–60 °C, Gln and ABA were added by filter sterilization (filter membrane pore size 0.22 μm). The medium was inoculated with MG as the explants and the cultures were cultured in the dark at 23 ± 2 °C.http://www.njjcbio.com/, accessed on 1 August 2022). Intracellular DHA content and DHAR activity were determined using Beijing Solarbio assay kits . The absorbance values of GSH and GSSG were determined by an enzyme marker, and the absorbance values of DHAR, ASA, DHA, GR, and APX were determined by spectrophotometry, according to kit instructions. Samples were prepared for each assay according to the following paragraphs. Methods were as follows:The intracellular GSH, GSSG, and ASA content, and GR and APX activity, were determined using Nanjing Jiancheng assay kits  for 10 min. The GSSG content was calculated according to the formula method. Three replicates per treatment were carried out. The concrete method please refer to the introductions.The concrete method please refer to the instructions.To determine ASA content: 0.2 g of EC was mixed with 0.5 mL of PBS buffer, homogenized in an ice bath, and centrifuged at 5000 rpm for 10 min. To determine DHA content: 0.1 g of the sample was mixed with 0.5 mL of extract, homogenized in an ice bath, and centrifuged at 16,000 rpm for 20 min at 4 °C. The absorbance value ‘A1’ was recorded at 10 s, and the absorbance value ‘A2’ was recorded at 130 s. The change (Δ) was calculated as A2-A1, and 0.5 μmol/mL DHA was used as the standard tube. Four replicates per treatment were carried out. The concrete method please refer to the introductions.To determine GR activity: 0.2 g of EC was mixed with 1.8 mL of PBS buffer, homogenized in an ice bath, and centrifuged at 2500 rpm for 10 min. Absorbance was measured at 340 nm with a 1 cm optical diameter cuvette. The absorbance value ‘A1’ was recorded at 30 s and the absorbance value ‘A2’ was recorded at 2 min at 37 °C. Double-distilled water was used as the standard tube. Four replicates per treatment were carried out. The concrete method please refer to the introductions.To determine APX activity: 0.3 g of the sample was mixed with 0.6 mL of buffer, homogenized in an ice bath, and centrifuged at 10,000 rpm for 10 min at 4 °C. The absorbance value ‘A1’ was recorded at 10 s, and the absorbance value ‘A2’ was recorded at 130 s. The change (Δ) was calculated as A2-A1. Double-distilled water was used as the standard tube. Three replicates per treatment were carried out. The concrete method please refer to the introductions.To determine DHAR activity: 0.6 g of the sample was added to 0.6 mL of extraction solution, homogenized in an ice bath, and centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was added to the reagent according to the method of the kit, and the absorbance value at 412 nm of the enzyme-labeled instrument was measured. Three replicates per treatment were carried out introductions.Transcriptomic sequencing revealed the presence of 10 differentially expressed genes between the F and S cell lines, and these genes were used for qRT-PCR validation. The Korean pine AAE7 gene  was used as an internal reference gene. The AAE7 gene was selected because it is stable, with gene activity being largely independent of external interference. Transcriptomic sequencing revealed the presence of 10 differentially expressed genes between the F and S cell lines, and these were used for qRT-PCR validation. Primer sequences were synthesized by Beijing Bemec Biotechnology Co., , and can be found in Experimental calculations, including proliferation parameters, were performed using Excel 2003 . A one-way analysis of variance (ANOVA) and Duncan’s multiple comparisons tests were performed using SPSS 19 . Data concerning percentages were arcsine-transformed before analysis. Graphs were constructed in Sigma Plot 12.0 . Proliferation parameters were calculated according to the following equations:The study confirmed that exogenous GSH promotes the proliferation of Korean pine EC. This is because the exogenous application of GSH increased the intracellular contents of T-GSH, GSH, GSSG, T-ASA, and ASA and the ratios of GSH: T-GSH and ASA: T-ASA in both F and S cell lines. It appears that the application of exogenous GSH promotes a reducing cultural environment, which is beneficial to EC proliferation in Korean pine. By helping to reveal the mechanism whereby GSH regulates redox homeostasis in Korean pine EC cells, we have laid the foundation for a large-scale breeding of Korean pine somatic embryogenesis technology system.


Cancer is a very challenging disease to treat, both in terms of therapeutic efficiency and harmful side effects, which continues to motivate the pursuit for novel molecules with potential anticancer activity. Herein, we have designed, synthesized, and evaluated the cytotoxicity of different brominated coelenteramines, which are metabolic products and synthesis precursors of the chemi-/bioluminescent system of marine coelenterazine. The evaluation of the anticancer potential of these molecules was carried out for both prostate and breast cancer, while also exploring their potential for use in combination therapy. Our results provided further insight into the structure–activity relationship of this type of molecule, such as their high structural specificity, as well highlighting the 4-bromophenyl moiety as essential for the anticancer activity. The obtained data also indicated that, despite their similarity, the anticancer activity displayed by both brominated coelenteramines and coelenterazines should arise from independent mechanisms of action. Finally, one of the studied coelenteramines was able to improve the profile of a known chemotherapeutic agent, even at concentrations in which its anticancer activity was not relevant. Thus, our work showed the potential of different components of marine chemi-/bioluminescent systems as novel anticancer molecules, while providing useful information for future optimizations. Cancer is one of the most challenging diseases currently faced by humanity; in 2018 alone, 17 million cases were reported worldwide, leading to 9.6 million deaths ,4. Even Clz)  that is usually overexpressed in tumor cells [Clz, with the formation of a high-energy cyclic peroxide intermediate (dioxetanone). This latter compound decomposes readily into the chemiexcited chemiluminophore, coelenteramide [Clz is also capable of bioluminescence in many marine organisms, in combination with either luciferase enzymes or photoproteins [Some members of this team have been active in recent years in the development of novel strategies for cancer therapy based on the chemiluminescent system of marine coelenterazine (Clz)  [6,7,8,9or cells ,13. Moreteramide ,11. Besiproteins ,11,14.Clz system to develop single-molecule and self-activating photosensitizers to overcome the current limitations of photodynamic therapy (PDT) [Specifically, our objective is to use the py (PDT) ,7,8,9. Ppy (PDT) ,16. In Ppy (PDT) ,16. Whilpy (PDT) ,18. So, Clz has potential to address the restrictions of PDT as the decomposition of the high-energy dioxetanone intermediate allows for the direct chemiexcitation from the singlet ground state to excited states [The chemiluminescent system of marine d states ,7,8,9, wd states ,11.Clz analogs (Cla compounds) [Cla compounds, Br-Cla  [In this context, members of this team have developed different single-brominated mpounds) ,7,8,9, wmpounds) ,7,8,9. Tmpounds) ,7,8,9. Mmpounds) ,11. Amon, Br-Cla  was initClz chemi-/bioluminescent system (Br-Cla included), with focus on their activity towards both gastric and lung cancer in vitro [Clm-1, Clz [Clz and analogs [Clm-1 showed higher anticancer activity than Br-Cla and other brominated Cla compounds, reaching IC50 values of 15.2 and 32.6 µM for gastric and lung cancer cell lines, respectively [Br-Cla and Clm-1  and breast (MCF-7) cancer cell lines. These were cell lines used previously in our initial evaluation of the anticancer activity of Br-Cla [Br-Cla and Clm-1 in cell lines other than those of lung and gastric cancer [Clm-1 analogs: Clm-2 and Clm-3  or 3,5-dibromopyrazin-2-amine (Clm-2) and an appropriated phenylboronic acids (either 4-bromophenylboronic acid or 3-aminophenylboronic acid). This type of reaction already yields both Clm-1 and Clm-3. For obtaining Clm-2, an additional reaction between N-bromosuccinimide and 5-(3-aminophenyl)pyrazin-2-amine (the product of the previous step) in ethanol is needed. The structures of compounds and synthesis intermediates were confirmed with both 1H/13C NMR and FT-MS spectroscopy  properties. Additionally, by analyzing their absorption/fluorescence in different media, we can observe how sensitive these compounds are to changes in the local microenvironment, and how differently they behave in relation to each other. This type of information could provide some potentially relevant insight, especially if we consider their mechanism of action as binding to an active/allosteric site of a yet-to-be-identified target.The next step of this study was to perform the photophysical characterization of 2O, MeOH, EtOH) and aprotic (ACN and DMF) media , and a ~20 nm red-shift to ~360 nm for the second. Once again, no relevant effect exerted by the solvent was found. Finally, Clm-3 does present more blue-shifted absorption, at both ~240–260 nm and ~310–320 nm, without any relevant effect from the solvent. Thus, these results indicate that only the structural modifications exert some effect on the UV-Vis absorption of these compounds. However, there is limited impact on the absorption wavelength maxima. The exception is just in DMF for Clm-2 and Clm-3.So, we started by measuring the UV-Vis absorption spectra of these compounds in protic (HF) media . All comClm molecules, the fluorescence emission spectra  and in H2O (by ~20 nm). In turn, Clm-2 is less sensitive to the solvent, and it presents an emission wavelength maximum of ~440–450 nm, irrespective of the solvent. A similar case is found for Clm-3, with emission maxima at ~440–450 nm. Interestingly, both Clm-2 and Clm-3 present a shoulder in emission spectra . Thus, these modifications do somewhat affect the properties of these compounds. It should be noted that, for Clm-1, there are some minor shifts in wavelength maxima from aprotic to protic solvents, and there are further shifts from MeOH/EtOH to H2O. This indicates some effect of hydrogen bonding on the fluorescence of Clm-1, which is not detected for the other compounds. Thus, Clm-1 might be able to participate in a different network of hydrogen bonding than the other compounds, which might be relevant in terms of binding to possible targets.As for individual fluorescence spectra , Clm-1 p spectra  in some Clm compounds, and how they actually differ from Br-Cla, we evaluated their in vitro cytotoxicity toward both breast MCF-7 and prostate PC-3 cancer cell lines. Namely, we evaluated their toxicity with increasing concentrations, while changes in cell morphology were observed by microscopy.To understand the anticancer activity of Cla compounds developed by us were active toward this cell line [Br-Cla presenting an IC50 of 21.6 µM [Br-Cla and other Cla compounds [First, we analyzed their effect toward MCF-7 breast cancer cells in terms of cytotoxicity  and effeell line ,7,8,9, wCla compounds showed activity toward this cell line [Br-Cla presenting an IC50 value of 24.3 µM [Clm-1 did show some activity by reducing the cellular viability by ~30 and ~50% at higher concentrations of 50 and 100 µM, respectively. By their turn, both Clm-2 and Clm-3 showed no anticancer activity. This is supported by microscopic analysis , in which was visible in the change in the size and shape of the cells, which were larger and longer. Compared with conventional chemotherapeutic drugs, such as doxorubicin or paclitaxel, we have previously demonstrated that these drugs have IC50 values of 0.17 µM [The next step was to evaluate the cytotoxicity  and chanell line ,7, with  24.3 µM ,8. Interanalysis , which r 0.17 µM  and 0.1 Clm-2/3 are clearly molecules without cytotoxic potential for normal cells, as they have no effect on the cellular viability (even at high 100 µM concentrations). However, the same cannot be said for Clm-1, in which a concentration of 100 µM induces toxicity of ~40% in MRC-5 cells. It should be noted that Br-Cla did not show any effect on the cellular viability of noncancer cells [Clm-1 has higher activity toward a noncancer cell line than for a cancer one (breast MCF-7), while having comparable activity toward prostate PC-3 cancer cell line. However, it should also be noted that the toxicity toward the noncancer cell line was only observed at a concentration (100 µM) relevantly higher than its IC50 toward both gastric and lung cancers (15.2 and 32.6 µM) [Clm-1 do show some potential as a compound worthy of optimization for future use in treatment of gastric and/or lung cancer.Finally, we also assessed the safety of this type of compounds, by evaluating their cytotoxicity toward noncancer MRC-5 cells. As presented in er cells . For one32.6 µM) . Thus, CClm-1-3 provide useful information for understanding the anticancer potential of the brominated Clm and Cla compounds. One of the conclusions is that despite their structural similarities, Clm-1 and Br-Cla present quite different behavior. That is, while Clm-1 is more potent toward lung and gastric cancer than Br-Cla [Cla is active (with variable efficiency) toward these four cancer types, Clm-1 is inactive toward breast cancer. Thus, these inconsistencies between the anticancer activity of these two compounds toward different cancer cell lines indicate that Clm-1 and Br-Cla should be considered as independent anticancer molecules with different mechanisms of action. This should mean that (i) Br-Cla can still be considered as a prototypical system for light-free and self-activating photosensitizers [Clz appear to be an interesting and unexpected source of different compounds with anticancer potential (when subjected to bromination).Despite the lack of relevant anticancer activity toward breast/prostate cancer cell lines, the results obtained for n Br-Cla , their rsitizers ,7,8, andClm-1 is particularly inflexible toward structural modifications. While our previous study identified the 4-bromophenyl moiety as essential to its activity [Clm-2) are enough to impair its anticancer activity. The same can be said by further substitution of the 4-bromophenyl moiety, by inclusion of additional bromine and amino groups, even while maintaining a bromine heteroatom at C4 (Clm-3). Thus, the mechanism of action of Clm-1 is clearly specific and sensitive to this 3-(4-bromophenyl)pyrazin-2-amine structure.The results toward both PC-3 and MCR-5 cell lines also allow us to conclude that the cytotoxic potential of activity , the preClm-1 (as the best-performing compound) in combination therapy [Clm-1 could affect the anticancer activity of the known chemotherapeutic agent 5-fluorouracil (5-FU) toward both MCF-7 breast and PC-3 prostate cancer cell lines, at the IC50 concentration of 5-FU (11.76 µM). Cellular viability was assessed with the standard MTT assay for an exposure period of 48 h, and the results are presented in Finally, we proceeded to test the potential of  therapy ,21,22. MClm-1 does not lead to relevant variations in cellular viability. This is expected, as this compound did not show anticancer activity toward this cancer type. However, the combination of this molecule with 5-FU does lead to some improvement in the profile of this chemotherapeutic agent. This is particularly interesting due to the lack of activity of Clm-1 and due to this improvement being seen already at concentrations as low as 1 µM (significantly lower than the concentration at which this compound is active toward noncancer cells).Starting with MCF-7 breast cancer cell lines , increasClm-1 does little for decreasing the cellular viability of the PC-3 cell line. However, there is now a somewhat relevant decrease at the higher concentration of Clm-1, which can be ascribed to its activity toward PC-3 cancer cells  or 3,5-dibromopyrazin-2-amine (Clm-2) and the corresponding arylboronic acid derivative using bis(triphenylphosphine)palladium(II) dichloride as the palladium source and potassium carbonate as the base. This reaction yields both Clm-1 and Clm-2. To obtain Clm-2, an additional halogenation reaction is required. Namely, the reaction between N-bromosuccinimide and 5-(3-aminophenyl)pyrazin-2-amine in ethanol is required. The structural characterization was performed by 1H NMR, 13C NMR spectroscopy, and FT-MS spectrometry. The results are available in the The synthesis of our team ,7,8,9, wClm-1-3 were measured with a Horiba Jovin Fluoromax 4 spectrofluorimeter, with an integration time of 0.1 s. Slit widths of 5 nm were used for both the excitation and emission monochromators. The absorbance spectra were measured using a VWR® Spectrophotometer (UV-3100PC) . Quartz cells with a 10 mm pathlength were used. The spectra were obtained in different solvents for each compound: acetonitrile ), N,N-dimethylformamide ), ethanol , methanol , and deionized water (H2O). All compounds were analyzed at a final concentration of 30 µM.The fluorescent spectra of 2. These reagents were purchased on Millipore Sigma . For maintenance, confluent cells were trypsinized using a 0.25% trypsin–EDTA solution  and subcultured in the same culture medium. The medium was changed every 96 h. Before treatments, 40,000 (MCR-5), 50,000 (MCF-7), and 25,000 (PC-3) cells/mL were seeded in 96-well plates and allowed to adhere overnight. Cells were used in passages 8, 45, and 15, respectively.MRC-5 human normal lung fibroblast cell line was used for evaluation of the biosafety profile of the compounds. MCF-7 human mammary carcinoma and PC-3 human prostate carcinoma cell lines were used for evaluation of the anticancer effect of the compounds previously synthesized. These cell lines were obtained from the American Type Culture Collection  and maintained in Dulbecco’s modified Eagle’s medium (DMEM) cell culture supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (pen–strep) solution, at 37 °C and 5% COClm-1, MCF-7, and PC-3 cells were treated with the IC50 of 5-FU (11.79 µM) and increasing concentrations Clm-1 , for 48 h. Controls were treated with 0.1% of methanol and 0.1% of DMSO (vehicles).The toxicological effect of each compound was evaluated after 48 h of treatment. Cells were treated with each compound in the following concentrations: 1, 25, 50, and 100 µM. Control cells were treated with 0.1% methanol (vehicle). For the evaluation of the toxicological effect of the combination of 5-Fluorouracil (5-FU) and The toxicological effect was evaluated by morphological analysis and MTT assay. Cell morphology was evaluated after each treatment using a Leica DMI 6000 B microscope equipped with a Leica DFCLM-350 FX camera . Images were analyzed with the Leica LAS X imaging software (v3.7.4) .The biosafety profiles and anticancer effects of each compound were evaluated by an MTT (thiazolyl blue tetrazolium bromide) assay. Briefly, after each treatment, cell media were aspirated and 100 µL of MTT solution  was added to each well; plates were incubated for 3 h at 37 °C in the dark. Subsequently, MTT solution was removed from each well and 100 µL of DMSO was added to each well to solubilize the formazan crystals. Absorbance was measured at 570 nm using an automated microplate reader . Cell viability was calculated as follows:n = 3). GraphPad Prism 9  was used to obtain the cell viability graphs. Results are represented as the mean ± SEM for n experiments performed. Statistical analysis was performed with one-way ANOVA tests by Dunnett’s multiple comparisons between control and treatment groups. Statistical significance was accepted at p values < 0.05.Experiments were conducted in triplicate  toward both breast and prostate cancer cell lines. Our objectives were to obtain further insight into the structure–activity relationship of these compounds, and to further clarify whether and how their mechanism of action is related with the family of brominated coelenterazine analogs with anticancer activity .Interestingly, we found that, despite their structural similarity (imidazopyrazinone core versus an aminopyrazine one), brominated coelenterazines and coelenteramines present quite distinct behaviors and potencies. Thus, these types of compounds should be considered as anticancer molecules with independent activities. We have also obtained evidence that the anticancer activity of brominated coelenteramines is structurally quite inflexible, being quite specific for the 3-(4-bromophenyl)pyrazin-2-amine structure. Finally, we also found that combining the best-performing coelenteramine analog improves the profile of a known chemotherapeutic agent, even at lower concentrations where its anticancer activity by itself is not relevant. Thus, it shows potential to reduce the dose required for chemotherapeutic drugs, while maintaining their efficiency.In summary, our results show that the chemi-/bioluminescent systems of marine coelenterazine are an attractive and unexpected source of different compounds with distinct and useful anticancer properties. Additionally, the obtained information should be useful in guiding future optimizations of these anticancer compounds.


Esophageal adenocarcinomais of increasing global concern due toincreasing incidence, a lack of effective treatments, and poor prognosis.Therapeutic target discovery and clinical trials have been hinderedby the heterogeneity of the disease, the lack of “druggable”driver mutations, and the dominance of large-scale genomic rearrangements.We have previously undertaken a comprehensive small-molecule phenotypicscreen using the high-content Cell Painting assay to quantify themorphological response to a total of 19,555 small molecules acrossa panel of genetically distinct human esophageal cell lines to identifynew therapeutic targets and small molecules for the treatment of esophagealadenocarcinoma. In this current study, we report for the first timethe dose–response validation studies for the 72 screening hitsfrom the target-annotated LOPAC and Prestwick FDA-approved compoundlibraries and the full list of 51 validated esophageal adenocarcinoma-selectivesmall molecules . We then focus on the most potentand selective hit molecules, elesclomol, disulfiram, and ammoniumpyrrolidinedithiocarbamate. Using a multipronged, multitechnologyapproach, we uncover a unified mechanism of action and a vulnerabilityin esophageal adenocarcinoma toward copper-dependent cell death thatcould be targeted in the future. This highlightsthe limitations of modern target-based drug discovery strategies toimpact upon complex heterogeneous diseases such as EAC. Phenotypicdrug discovery describes the screening and selection of compoundsbased on quantifiable phenotypic end points without prior knowledgeof the drug target.11 It is therefore an attractivestrategy for heterogeneous diseases, where there is a lack of understandingof disease biology and actionable targets.Whole genome sequencingof clinical samples has recently highlightedEAC as highly heterogeneous, characterized by frequent large-scalegenomic rearrangements and copy number alterations.13 The Cell Painting assay has recently beenused to screen 30,616 small-molecule compounds in the U2OS osteosarcomacell line, generating a large repository of compound phenotypic fingerprintsto support the development of new methods, including artificial intelligence/machinelearning approaches, which associate cell phenotypes with chemicalstructures.14Cell Painting isa high-content phenotypic screening assay thatmultiplexes six fluorescent probes labeling multiple cellular compartments.When combined with image analysis and computational biology tools,the Cell Painting assay generates a phenotypic fingerprint for everycell, following chemical or genetic perturbation to identify chemicalstarting points and novel targets and help guide mechanism-of-actionstudies.15 We report forthe first time the full list of primary screening hits and dose–responsehit validation studies from the target-annotated LOPAC and PrestwickFDA-approved compound libraries. Further, we characterize the mostpotent and selective hit compounds ) in a holistic approach to understandthe molecular mechanisms that confer drug sensitivity and potentiallyidentify new therapeutic targets and classes of small molecules forthe treatment of EAC.In the current study,we have undertaken a comprehensive small-moleculephenotypic screen using the Cell Painting assay to quantify the morphologicalresponse to a total of 19,555 small molecules across a panel of sixgenetically distinct human EAC cell lines and two nontransformed tissue-matchedcontrol cells to identify new therapeutic targets and small moleculesfor the treatment of EAC.22 Elesclomol has been reported to act as a reactive oxygen speciesinducer,22 while disulfiram is an alcoholdehydrogenase inhibitor,19 and ammoniumPDTC is a nuclear factor kappa B (NFkB) inhibitor.17 Disulfiram and ammonium PDTC are also reported to affect the proteasomevia multiple mechanisms, including contradictory reports of proteasomeinhibition23 and NLP4 aggregation21—a key component of the p97/VCP segregaseessential for protein turnover. Despite differing and at times conflictingreported activities, structurally, the compounds share similaritiessince all three contain thiocarbonyl groups, and they are known copperchelators. Ammonium PDTC is part of the family of dithiocarbamates,while disulfiram, a thiuram disulfide, breaks down in acidic or Cu(II)-richenvironments to produce a dithiocarbamate, diethyldithiocarbamate(DDTC).24 DDTC and ammonium PDTC, likeother dithiocarbamates, are known to form complexes with transitionelements but are most stable in a Cu(II) chelate.25 Although elesclomol is structurally unrelated to the dithiocarbamates,it also forms organometallic complexes, particularly with Cu(II),due to its dihydrazide and thiocarbonyl groups.27Previous studies have indicated that thesethree compounds havevaried targets and therefore likely act via multiple distinct mechanisms.In this work, we employ a multipronged, multitechnology approachand uncover a unified mechanism of action shared by all three compounds.We believe the holistic approach we have employed in this study supportsthe identification of hit compound mechanisms of action that targetcomplex heterogeneous diseases with high selectivity.15 (Supporting Table 1) as dose responses acrossa panel of heterogeneous esophageal cell lines. The dose–responsevalidation was performed across two phenotypic assay end points: 1.nuclei count to quantify cell survival and 2. multiparametric morphometricresponse (quantifying 702 features) to provide a more in-depth andunbiased analysis of phenotypic response to compound treatments.The aim of thisstudy was to perform an unbiased and target-agnostic phenotypic screento identify potential drug repurposing opportunities, new chemicalstarting points, and targets to stimulate drug discovery in the challengingdisease area of esophageal cancer. To uncover the mechanism of actionof phenotypic hits, which display high selectivity for EAC lines relativeto nontransformed tissue-matched controls, we have generated multiparametriccellular phenotypic fingerprints for each compound. For validatedcompound hits, which display high sensitivity and selectivity forEAC cell lines, we link compounds that share similar phenotypic fingerprintsand transcriptomic profiles to the chemical structure to further elucidatethe compound mechanism of action. In this work, we began by performingimage-based phenotypic profiling of 72 compound hits identified fromour primary phenotypic screenSupporting Table 2, and Supporting Data). Three of the selectivecompounds  were initially identifiedas active in the multivariate primary analysis but not the cell survivalanalysis, demonstrating the utility of morphometric phenotypic screeningto detect phenotypically active compounds that would otherwise goundetected from univariate survival analyses of single-point concentrationsat the primary screening stage.Testing the 72 compounds for dose-dependent cell survival, we foundthat 47 of the 72 compounds  reduced cell survivalin a dose-dependent manner, of which 11 (15%) showed strong selectivityfor the EAC cells over the tissue-matched control lines (Supporting Table 2). Across thetwo end points, we identified 51 small molecules that showed dose-dependent activity across two or more of the EACcell lines.In the unbiased phenotypic analysis, we also developeda multivariatemorphological dose response to validate morphological changes overa range of concentrations from maximal to minimal effect. Here, 51of the 72 total compounds (71%) caused dose-dependent phenotypic changes,of which 46 (64%) were selective for the EAC cells over the tissue-matchedcontrols , that compounds with a shared mechanismof action cluster phenotypically.15 Thephenotypic dose responses for the validation hits demonstrate class-specificphenotypic clustering , followed bydisulfiram and then ammonium PDTC. Critically, the dose responsesshow no toxicity in either of the tissue-matched nontransformed controlcell lines  and both continued to proliferateat their normal rate . These data point to a novel form ofcell death, cuproptosis, a recently proposed mechanism of cell deathspecific to copper.30We then chose to follow up the most potentand selective small molecules identified across our assay panel—elesclomol, disulfiram, and ammonium PDTC . Elesclomol was also highly selective, inhibitingthe viability of tumor-derived organoids and not the normal gastricorganoid NG088  to better understand how the compounds affect thecells within the context of known drug mechanisms of action. The phenotypicdose responses show that both disulfiram and ammonium PDTC move fromphenotypically inactive at low concentrations, clustering with thedimethyl sulfoxide (DMSO), to phenotypically active at higher concentrations, further demonstratingstrong selectivity.Using the phenotypicinformation from the Cell Painting concentrationresponses, we built multivariate dose responses, tracking cellularmorphological changes as a product of compound concentration. We overlaidthese dose responses on a library of reference compounds we have publishedpreviouslytrations2D, whiletrations2D. Of no22 However, our data indicate that all three compounds belong to thesame morphological cluster. Quantification via correlation of themultivariate phenotypic signatures confirmed all three compounds inducea shared morphological response  , suggesting a potentiallyshared mechanism in EAC. Furthermore, studying the univariate cellsurvival analysis, the compounds also shared the same sensitivityprofile across the panel of cell lines, with OAC-P4C showing the greatestsensitivity and FLO-1 showing the weakest   , demonstrating that disulfiramand elesclomol induce the same gene expression signature. Interestingly,neither elesclomol nor disulfiram induced any significant gene expressionchanges in the two tissue-matched controls .To further confirm ashared mechanism, we examined compound-inducedgene expression signatures. Comparison of signatures can be used todiscover new connections among compounds as well as to identify targetsand mechanisms of action in a more complex setting than single-genecontributions.lllines 3C.Seconlllines 3C. PearsSupporting Figure 6). We therefore do not believethat disulfiram, elesclomol, and ammonium PDTC are targeting the sensitiveEAC lines via ALDH inhibition.Disulfiramis a well-definedALDH inhibitor; to rule out this as the mechanism in EAC cells, threeALDH inhibitors were tested for cytotoxicity across esophageal celllines. CVT-10216, a potent and selective reversible inhibitor of ALDH2, and two potent ALDH1A1 inhibitors (NCT-501 andA37) were tested, none of which had any effect on cell viability (upto 10 uM) in either the tissue-matched control or EAC cell lines  to remove free copper from the media led to complete lossof activity for both disulfiram and elesclomol  were unable to rescuecell death, demonstrating specificity for copper.Disulfiram, elesclomol, and ammoniumPDTC are also known to form organometallic complexes with copper25 4A, and iesclomol 4B, confiSupporting Figure 7 for Fe levels and data not shown for other metal ions), againdemonstrating the specificity for copper in their mechanism of action.Consistent with the lack of cytotoxicity and gene expression changes,ICP-MS showed no accumulation of copper in the tissue-matched controlcells after incubation with either disulfiram or elesclomol (Using inductively coupled plasma mass spectrometry (ICP-MS) todetect metal ions within cell extracts, we found that both disulfiramand elesclomol treatment led to the accumulation of intracellularcopper in EAC cells 4C, ident35 revealed strong induction of multiple heat shock, growtharrest and DNA damage genes, and heme oxygenase I .Given the clear variability in EAC cell line sensitivity to disulfiramand elesclomol, we sought to identify the mechanisms that confer sensitivitythrough the integration of basal transcriptomic data, as this mayfurther elucidate the mechanism through which they act. Given thatTP53 mutations are the most frequent alteration in esophageal cancerreatment 5C, we ex37 for seven EAC cell lines to revealsignificant associations at the biological pathway level. GSEA ofhallmark gene sets revealed 10 that positively correlated and 11 thatnegatively correlated with IC50 (p <0.05) (Supporting Table 6). In concordance with this, the unfolded protein responseis the fifth gene set identified. Furthermore, GSEA of Kyoto Encyclopediaof Genes and Genomes (KEGG) gene sets revealed 18 that were significantlyenriched and positively correlated with elesclomol sensitivity, ofwhich the proteasome was the top gene set  to publicly availablegene expression data <0.05) 6A.MYC tgene set 6B.Supporting Figure 9). MSRB genes have also beenstudied in relation to copper overload in yeast and endoplasmic reticulum(ER) stress previously.40 Follow-up work is required todetermine the contribution of these biomarkers to copper-induced celldeath in this setting.We have also carried out differentialexpression analysis of nineEAC lines profiled with the NanoString nCounter platform and a pancancer correlation of gene expression using the Genomics of Drug Sensitivityin Cancer data to identify potential sensitivity biomarkers at thegene rather than pathway/network level. Preliminary results suggesta number of potential biomarkers of sensitivity; we highlight methioninesulfoxide reductase B2 (MSRB2), B3 (MSRB3), and glutathione peroxidase8 (GPX8) as this pathway is consistent across both analyses (41), ubiquitin andproteasome subunit expression correlating with sensitivity, and thefact that copper is known to bind proteins and induce misfolding andthe unfolded protein response,44 we utilized the morphologicaldata to study potential links to protein misfolding and proteostasisto further elucidate the mechanism by which these compounds work.Given the identification of upregulatedheat shock response andunfolded protein response after compound treatment  .< 0.001) 7C, altho47 though we believe this is not via targeted delivery but by simplybypassing the defunct copper transport system in copper disordersand increasing bioavailable copper levels within cells.Overall, using a panelof cell lines, we applied the Cell Paintingassay to identify and cluster selective hit compounds for the treatmentof EAC. Then, through the integration of morphological and transcriptomicdatasets, we began to deconvolute the mechanism of this cluster ofEAC selective compounds, elesclomol, disulfiram, and ammonium PDTC,and identified a unified mechanism of action. We provide evidencetoward a mechanism and vulnerability of EAC cells involving an influxof intracellular copper accumulation leading to dysregulation of proteostasisand cancer-specific cell death. This is in line with studies of copperdeficiency disorders that demonstrated elesclomol is capable of restoringcuproenzyme activity and alleviating disease symptoms,41 similarityin gene expression signature to known proteasome inhibitors, and identificationthat proteasome pathway basal gene expression levels predict sensitivityto elesclomol and disulfiram from GSEA analysis. Crucially, giventhe dissimilarity in morphological signatures and opposing sensitivityof the copper ionophores and known proteasome inhibitors across theEAC panel, we believe that these copper ionophores act via a mechanismthat is distinct from the proteasome inhibitors while still inducingthe same downstream response—loss of proteostasis. This fitswith the finding that disulfiram blocks the cellular machinery involvedin misfolded protein response by causing the aggregation of NPL421 but does not affect the CT-like, C-like, orT-like activity of 20S proteasome, and recent findings that elesclomolcauses the aggregation of lipoylated proteins.30 In this current work, we bring these separate observationsfor disulfiram and elesclomol together by suggesting that proteinaggregation is a nonspecific and global response to copper ionophoresand is not due to the aggregation of any one specific protein target.Further, our conclusions fit with recent data demonstrating that copperions interact with proteins to impair folding and promote proteinaggregation.44Evidencetoward a mechanism resulting in dysregulation of proteostasisis provided through the identification of a compound-induced geneexpression signature involving pathways known to arise from increasedlevels of misfolded proteins,48 Since links between the proteasome and cellularmetabolism have been established in other areas of research,50 we do not believe our current findings relating to elesclomol andproteostasis are mutually exclusive from those relating to metabolism.In our work, we do note the identification of multiple metabolic pathways,including oxidative phosphorylation, fatty acid metabolism, glycosphingolipidbiosynthesis, and bile acid metabolism, in our copper ionophore sensitivityGSEA . Future studiesto fully understand the role of metabolism and mitochondrial respirationand its links to proteostasis in the specific context of copper ionophoresmay shed further light on the mechanisms at play.While not the focus ofour work presented here, we do acknowledgethat a role for metabolism and mitochondrial respiration in elesclomolsensitivity has been suggested in previous work.ityGSEA 6. FurtheIn conclusion,this work provides a model framework to identifyand deconvolute new therapeutic targets and classes of small moleculesutilizing a multipronged, multitechnology, holistic approach. We believethe holistic and target-agnostic approach we have employed in thisstudy across panels of cell lines embraces the complexity of heterogeneousdiseases and can be applied early on in the drug discovery pipelineto have a beneficial impact upon drug discovery success rates as awhole in such disease areas of unmet need.l-glutamine .CP-A and EPC2-hTERT cells were grown in KSFM with 5 g L–1 human recombinant epidermal growthfactor and 50 mg L–1 bovine pituitary extract (Supporting Table 7 for cell line details).EAC lines were grown in RPMI-1640  with 10% fetal bovine serum (FBS)  and 2 mM 29 Cells were seededin a complete medium and then treated with elesclomol in 9-point half-logserial dilution for 6 days . Treatmentswere performed in technical duplicates and at least two biologicalreplicates. Cell viability was assessed using CellTiter-Glo (Promega).Primary organoid cultures were derived from normal gastric and EACcases included in esophageal cancer clinical and molecular stratification(OCCAMS)/international cancer genome consortium (ICGC) sequencingstudy. Detailed organoid culture and derivation method have been previouslydescribed in detail.Cells were seeded at 800–1500cells per well in 50 μL into 384-well microplates  for 24 h. Compounds were then added as 8-point semi-log doseresponses from 10 μM before being incubated for a further 48h.13 Cells were fixed in 4% formaldehyde before permeabilization in 0.1%Triton-X100 (v/v). Finally, the staining solution was added in 1%bovine serum albumin in PBS (w/v) and incubated for 30 min beforebeing washed out. (Supporting Table 8).Four fields of view were captured per well using a 20× objectiveand five filters (Supporting Table 8).For morphological and nuclei count readouts, the Cell Paintingprotocol was applied, which uses multiplexed fluorescent dyes to visualizecellular and subcellular organelle and cytoskeletal morphology.For the expanded panel of EAC cell lines, data CellTitre-Glo (promega)was added according to the manufacturer’s instructions andincubated for 10 min at room temperature before being read. For caspase-dependentapoptosis assays, we monitored caspase activity at sequential timepoints following compound treatments using the Incucyte imaging instrumentand caspase-3/7 biosensor according to the manufacturer’s instructions(Sartorius).CellProfiler v3.1.5software was usedto extract features from the images. The cell-level data was aggregatedto the image level by taking the median for each measured featureper image. Low-quality images and image artifacts were then identifiedand removed using image quality metrics extracted by CellProfiler.Images with 20 cells or less were also removed from the dataset. Forthe remaining images, features were normalized on a plate-by-platebasis by dividing each feature by the median DMSO response for thatfeature. Features with NA values were removed, as were features withzero or near-zero variance, using the “findCorrelation”and “nearZero” functions in the R package Caret. Allremaining features were scaled and centered globally by dividing bythe standard deviation of each feature and subtracting the featuremean, respectively. The pairwise correlations were calculated forall remaining features, and highly correlated features (>0.90)wereremoved. Finally, the image data was aggregated to the well (compound)level, and this was used in the analysis.http://www.r-project.org using software packages available via CRAN http://cran.r-project.org andBioconductor http://www.bioconductor.org.All analysis was conductedin R Copper chelator bathocuproinedisulfonic acid (BCS). The doseresponseswere carried out as stated for the validation dose responses, exceptthat disulfiram and elesclomol dose responses were carried out inthe presence or absence of 200 μM BCS preincubation for 15 min.4 cells in 6-well plates for 24 h. Media was replacedwith fresh media containing compound treatments (DMSO (0.1%), disulfiram(600 nM), or elesclomol (200 nM)) before further incubation for 6h. Media was then removed, and plates were washed twice with ice-coldPBS before being snap-frozen at −80. Cells were scraped andlysed using QIAshredders  and Qiagen RNeasy Mini kit (with β-mercaptoethanol) according to the manufacturer’sinstructions and included a DNase digestion step .Briefly, 100 ng of each sample was loaded into the NanoString nCounterAnalysis System with the Human PanCancer Pathways and Metabolic Pathwayspanels. Raw counts were normalized to the internal positive controlsand housekeeping genes using the nSolver 4.0 software. nSolver 4.0software and the NanoStringDiff algorithm35 were used for differential expression analysis. P-values were adjusted using the Benjamini–Yekutieli approach.54 Treatment-induced analysis was carried out betweenthe control (DMSO) and treatment (200 nM elesclomol) samples for thetwo most sensitive cell lines (OAC-P4C and SK-GT-4) pooled together. N = 3. The difference in cell lines was taken into accountas a confounder in the analysis.Cells were seeded at 8 ×106 cells were seeded in a T175 flask and incubated for24 h. Media was replaced with compound treatments (DMSO (0.1%), disulfiram(600 nM), or elesclomol (200 nM)) before further incubation for 6h. Cells were washed in PBS, trypsinized, and counted. For each sample,2 × 106 cells were pelleted and frozen at −80°C. For analysis, samples were thawed, and concentrated nitricacid was added (100 μL) and mixed. Samples were then vortexedand sonicated and left overnight at room temperature. Samples weremade up to 1 mL using water and then further diluted 5-fold priorto analysis of copper content by inductively coupled plasma mass spectrometry(ICP-MS).Typically, 5 ×1051 on the genomicsof drug sensitivity in cancer Robust Multichip Average (RMA) processedgene expression data  for thecell lines FlO-1, OE19, OE33, OAC-M5.1, OAC-P4C, SK-GT-4, ESO26, ESO51,and our own IC50 sensitivity data for elesclomol, usingthe H and C2 gene sets from the molecular signatures database . Default settings were used, except that Pearsonwas used for gene ranking.Enrichment analysis wasconducted using GSEA,52H-Hallmark gene set collection includes50 gene sets.53C2-Canonical pathwaysKEGG collection includes 186 gene setsGDSC1000 downloads:http://www.cancerrxgene.org/gdsc1000//Data/preprocessed/Cell_line_RMA_proc_basalExp.txt.zip.The Robust Multichip Average (RMA)-processeddataset is availableat


The effectiveness of chemotherapy in cancer cell regression is often limited by drug resistance, toxicity, and neoplasia heterogeneity. However, due to the significant complexities entailed by the many cancer growth processes, predicting the impact of interference and symmetry-breaking mechanisms is a difficult problem. To quantify and understand more about cancer drug pharmacodynamics, we combine in vitro with in silico cancer models. The anti-proliferative action of selected cytostatics is interrogated on human colorectal and breast adenocarcinoma cells, while an agent-based computational model is employed to reproduce experiments and shed light on the main therapeutic mechanisms of each chemotherapeutic agent. Multiple drug administration scenarios on each cancer cell line are simulated by varying the drug concentration, while a Bayesian-based method for model parameter optimisation is employed. Our proposed procedure of combining in vitro cancer drug screening with an in silico agent-based model successfully reproduces the impact of chemotherapeutic drugs in cancer growth behaviour, while the mechanisms of action of each drug are characterised through model-derived probabilities of cell apoptosis and division. We suggest that our approach could form the basis for the prospective generation of experimentally-derived and model-optimised pharmacological variables towards personalised cancer therapy. Clinical development of anti-cancer drugs is a resource-intensive and time-consuming process, which requires effective novel preclinical platforms for screening compounds targeting neoplasia . The theIn addition to the experimental methods, theoretical approaches have experienced an uptake in medicine, as they help simplify and conceptualise underlying biological complexities. Indeed, concepts such as symmetry and symmetry-breaking are employed also to formalise cancer dynamics ,6. NotabAt the tissue or organ scale, physics-based in silico models often employ continuum-based  biomechanics governed by partial differential equations. Such models have been reported to simulate avascular or/and vascularised tumour growth, biofluid flow and drug transport phenomena. Relevant to the above, pharmacokinetics/pharmacodynamics-based models have been able to simulate the exchange of drugs among compartments and accumulation of drugs within a particular organ or the cancerous tissue .Another category of in silico models in biomedicine that has been receiving increased popularity is that of spatially discrete, particle-based models which describe entities as agents, hence referred to as agent-based models . The majIn this manuscript, we propose a combined in vitro/in silico approach to evaluate and characterise the anti-tumoural activity of commonly used chemotherapeutic agents. The in vitro experiments we conducted report on the effects of selected cytostatics on two established cancer cell lines  through pertinent cytotoxicity laboratory tests. The in silico modelling framework we developed combines off-lattice agent-based modelling and a Variational Bayesian Monte Carlo method to simulate cancer cell culture development, with or without the presence of a cytotoxic agent. The in vitro evidence is used within the computer-based modelling procedure for estimating the probabilities of cell division and apoptosis for each treatment scenario, thus providing insights into the action of each chemotherapeutic drug. Our work contributes to cancer research by reporting a new combined in vitro/in silico methodological approach for cancer drug screening through the quantification of cytotoxicity effects on cancer cell proliferation and apoptosis. The paper starts with the methodological description of the in vitro experiments and cytotoxicity analysis, and continues with the presentation of the in silico framework, including ABM and model parameter optimisation. Subsequently, we report on the experimental results of the cytotoxicity laboratory tests, the computational model assessment and verification, and the corresponding model-derived evaluation of the effect of the different treatment regimes. Important findings of this work are summarised in 2 culture flasks. The cells were grown in an incubator in a humidified atmosphere with 5% CO2 at a physiological temperature of 37 °C, and, after a few passages and a confluence of about 80%, the cells were analysed with the MTT assay. We examined cytotoxic and thus anti-proliferative activity of selected cytostatics on two model systems: a human colorectal carcinoma HCT-116 cell line and a human breast cancer MDA-MB-231 cell line. The cell lines of low passages were acquired from the European Collection of Authenticated Cell Cultures and were maintained in Dulbecco’s Modified Eagle Medium (DMEM)  supplemented with 10% foetal bovine serum  and 1% penicillin/streptomycin  in 75 cmThe capability of tested cytostatics to reduce the cell growth of two different cell lines was assessed by a standardised MTT assay (Laboratory for Bioengineering protocol CB-005). An approximate 10,000 cell population per well (in 96-well microplates) were seeded and cultured in an incubator for 24 h to allow cell adhesion. After incubation, the cells were treated with cytostatics—the cytostatics used in this study are commonly used in clinical practice. For the colon cancer treatment, 5-Fluorouracil, Oxaliplatin, Leucovorin, and Irinotecan were used, while, for the breast cancer treatment, Endoxan, Paclitaxel, Docetaxel, and Doxorubicin were used. The cytostatics/antibodies were kindly donated by the University Clinical Centre of Kragujevac, Serbia. The chosen concentration range was standardised from 0.1 to 500 le cells .IC50 values were calculated from the dose curves using the CalcuSyn software. These values represent the amount of the substance required in order to decrease the number of cells by 50%. In principle, an anti-cancer drug is anticipated to express a clear selectivity towards cancer cells. Thus, the in vitro screening permitted us to measure drug- and cancer (cell line) specific effects—the experimental measurements yielded IC50 value estimates, expressed in molar concentrations.Cytotoxic activity was experimentally interrogated in six repetitions for each treatment dose scenario. Statistical analyses were evaluated by using the one-way ANOVA test for multiple comparisons by employing the SPSS statistical software package . The Our in silico approach employs a probabilistic agent-based model (ABM) designed to recapitulate our in vitro experiments and help understand the dynamics of cancer cell responses to treatment. In principle, ABM is a complex-system modelling method that assumes autonomous and interactive ‘agents’. Each agent is denoted a particle positioned in space following an off-lattice modelling approach. This work assumed a two-dimensional space, albeit ABM can generalise in three dimensions seamlessly. Thus, agents were set to freely move in a disk plane (diameter 2.4 mm) under specific restrictions for agent–agent overlap and collapse, while we assumed that the boundary of the simulation domain extends periodically to encompass the entire in vitro well. The behaviour of each agent was determined by simple rules and interactions with other agents, as well as external stimuli: the cytotoxic drugs in this work. These individual rules and interactions created patterns, structures, and produced time-varying outcomes for the entire biological system, which was not explicitly programmed into the model.P• denotes the probability of occurrence for a cell biological process. Presently, the probability for cell migration and cell growth was set fixed to 99% and 90%, respectively, whereas the probability for cell apoptosis and cell division (either programmable or induced by the drug) were the subject of investigation in the parametric analysis that follows in the 2 saturation level on a fixed average rate of 0.1 Algorithm 1 Cell behaviour algorithm that is iterated for all cells (agents) and for all time-steps of an ABM simulation.if random_number then      Remove cell from simulationreturn      if random_number then      Displace cellif random_number thend, unless       Develop cell diameter, return      if random_number then      Create new copy of (mother) cell: (daughter) cell      Half the volume of (mother & daughter) cellsIn this study, agents represented individual cells that were considered as being either of the human colorectal carcinoma HCT-116 or of the human breast cancer MDA-MB-231 cell line. The unknown parameters of our ABM simulations were estimated using the Variational Bayesian Monte Carlo (VBMC) method . VBMC beapproach .Papoptose, and the probability of cell division, Pdivide. Gaussian distributions were used as priors for both parameters, while the prior means were set to Papoptose) = 0.2 and Pdivide) = 0.15, respectively. Parameters were estimated by fitting the in silico model-derived cell viability to the relevant in vitro values.The VBMC framework has been employed within this work to efficiently estimate approximate posterior distributions and lower bounds to the model evidence of our models. Specifically, the two unknown ABM parameters for each treatment scenario were estimated numerically, namely the probability of cell apoptosis, C++ project invitro_neuro that was built and maintained on Bitbucket. A snapshot of the simulator can be downloaded from Figshare. Link to access the computer code from Figshare: https://doi.org/10.6084/m9.figshare.19387094.v1. It employed at its core the open-source software platform BioDynaMo (version 1.1.70). BioDynaMo [vbmc that is coded in MATLAB. All in silico experiments were performed on a MacBook Pro  with a 2.3 GHz 8-Core Intel Core i9, and 64 GB (2667 MHz DDR4) memory. Each individual ABM simulation run (using our in-house project invitro_neuro was executed using two threads in a Bourn-again shell) required approximately 2—3 s, whereas each batch of a full optimisation algorithm run  required 342.1 ± 36.6 s.The agent-based modelling method for this work was implemented in a ioDynaMo  was usedIC50 values) of the cytostatics tested on each cancer cell line respectively after 24 and 72 h of exposure to the drug.Cytotoxicity in vitro experiments were performed to investigate the biological influence of selected commercial cytostatics against colorectal carcinoma HCT-116 and breast adenocarcinoma MDA-MB-231 cell lines. In IC50 values , Oxaliplatin (Ox-Pt), and Irinotecan after 72 h, while Leucovorin exerted a marginal cytotoxic effect at low to moderate drug concentrations see also . Besides0 values a revealehttps://figshare.com/projects/In_vitro_cancer_drug_pharmacodynamics_via_Agent-based_and_Bayesian_Monte_Carlo_modelling/133802 (accessed on 20 February 2022).Next, the in silico modelling procedure was tested and verified against the in vitro data of the cytotoxicity assay experiments. Comparing the in vitro, blue lines, to the in silico results, red lines, the model demonstrates its capacity to capture the experimentally measured cell viability very well. Indeed, the in vitro viability (for both cell lines and at most cytostatics) demonstrated a decline with increased drug concentration, see p-value. The values to these quantities are listed in The model accuracy was also quantified by assessing the correlation of the in vitro and in silico cell viability through a linear regression model. Model accuracy was assessed by calculating the Papoptose and Pdivide, were varied by Papoptose = 17.5% and Pdivide = 15.65%, while, for the MDA-MB-231 control, the parameter values were Papoptose = 19.34% and Pdivide = 17.36%.To assess the accuracy and reliability of the in silico model, we quantified the impact of selected model parameters on the final number of cancer cells  by considering the untreated case scenario (control) for both cell lines. Parameter sweeps were performed for assessing the behaviour of model parameters, whereby the apoptosis and division probability parameters of the agent-based model, Papoptose and Pdivide, respectively. A scaling factor of 1 for the apoptosis and division probabilities corresponds to the baseline pair of parameter values adopted in the control simulations, as demonstrated in the verification tests in Papoptose and Pdivide produce relatively small changes to the simulation results. Therefore, the pattern of the absolute error distribution  in Papoptose lead towards the cell population decline—if a cell dies, then it cannot contribute to the overall cell growth regardless to its propensity to divide and create new cancer cell copies.Pdivide, and apoptosis, Papoptose. In order to circumvent any biases due to differences between the various treatment experiments, the probabilities of division and apoptosis were normalised with respect to their corresponding control values for each drug screening experiment: P*divide = Pdivide(Treated) /Pdivide(Control), and P*apoptose = Papoptose(Treated) / Papoptose(Control). Original, non-normalised values for the apoptosis and division probabilities are provided in Following the in silico model verification tests, we employed the experimental data of the cytotoxic agents for both in vitro model systems  within our modelling framework to further assess drug cytotoxicity effects. The mechanisms of action of each drug were examined through the model-predicted probabilities of cancer cell division, P*divide / P*apoptose. In principle, values of the survival probability ratio close to 1 could either signify no significant deviations from the control values or correspond to similar changes in the probabilities of division and apoptosis—the latter scenario being less probable as a drug would likely act by increasing apoptosis and decreasing division and vice versa. On the other hand, values of cancer cell survival below 1 denote a stronger anti-tumoural effect.Due to the coupling between the probabilities of division and apoptosis see , a new it-test with unequal variance was employed to test the hypothesis that the mean of cancer cell survival for the low-concentration drug is lower than the mean of the high-concentration drug. Statistically significant results  are depicted with asterisks in In view of the in silico HCT-116 results demonstrated in The in silico MDA-MB-231 results in Computational models are increasingly involved in biomedicine and pharmaceutical development. One of the main reasons for this is that in silico methods offer a complementary approach to conduct research and development, in addition to wet-lab experimental work and purely analytical techniques. Indeed, such a precision medicine approach offers numerous benefits such as lower costs, reduced need for animal experiments, or the potential for highly adaptive and individualised patient care. In particular, it constitutes an attractive method to deal with complex symmetry-breaking processes, which play a crucial role during cancer progression ,22. We tFolfox protocol represents treatment with Ox-Pt, Leucovorin, and 5-FU on the first and second day. This combination administered on these two days represents Cycle 1. The next cycle is applied after 14 days. The number of cycles depends on clinical evaluation. Another example is protocol Folfiri, which represents a combination of Irinotecan, Leucovorin and 5-FU in the very similar fashion as in the Folfox protocol. The synergism of applied drugs is crucially important for patient outcome. Similar to colon cancer, breast cancer patients also undergo protocol-based therapy. An AC protocol represents a combination of Doxorubicin with Endoxan administered once every three weeks. Paclitaxel and Docetaxel are used as single therapeutics administered in weekly intervals prescribed by the medical expert. We are currently conducting in vitro experiments of combinations of chemotherapeutic drugs, with preliminary results indicating synergistic drug effects in cancer regression. As such, we plan to carry out a large-scale parametric analysis study, using our in silico method, to interrogate and optimise combinatory chemotherapeutic protocols.Our computational results complement our experimental cancer model results. In particular, we find that well-established chemotherapeutic drugs have a differential impact on fundamental tumourigenesis parameters—in particular, the ratios between proliferative and apoptotic activities. We suggest that this ratio can become a quantitative measure of cancer progression and viability with a prognostic potential. For instance, drugs could be chosen in a context-specific manner, taking into account indicators of the proliferative rate of a patient’s cancer vs. the propensity to migrate and metastasise. Our computational results also indicate that the effects of these drugs significantly differ with regard to the concentrations at which they should be administered. Despite the fact that this study focused on a specific drug mode of action , the proposed in silico ABM methodology can be readily extended to account for other combinations of cytotoxic effects, e.g., reduce cell invasiveness, down-regulate differentiation to metastatic phenotypic behaviours, etc. However, quantitative measures of these aspects can be inferred by combining ABMs with wet-lab experiments, accelerating hypotheses’ generation and testing. From a purely experimental perspective, the treatment of colorectal and breast carcinoma cells revealed that not all cytostatics exert strong anticancer activity, even though these drugs are commonly used in clinical practice. This is not surprising because the protocols for patient treatment include the use of combinations of these drugs depending on the type and stage of the cancer. For example, in the treatment of colon cancer, the Notably, our approach has demonstrated in this paper a moderate computational footprint, which offers the prospect of exploring a broad model parameter space . Therefore, it further supports the usage of ABM for determining the most effective treatment schedules or strategies. For instance, the in silico can be used to generate causal predictions on the efficacy of different treatment durations, or scheduling protocols that are not present in the initial training data.Equally important to incorporating in vitro experimental data is the capability to take into account other background knowledge on cellular metabolism and drug interactions that can be seamlessly incorporated into our mechanistic model. This distinguishes our approach from other computational techniques, such as deep neural networks, where statistical models are generated from the training dataset. Such ‘black box’ approaches do not mechanistically model biophysical processes, and so it is not possible to include previously established rules, or extrapolate scenarios that are not implicit in the training data. In contrast, our approach allows for generating verifiable hypotheses also for treatment scenarios not captured by the experimental data.Last but not least, a particularly promising avenue is to employ our approach to interrogate and test candidate drugs and tailored combinations of drugs that will likely have the most beneficial impact in killing specific cancers. In this regard, they constitute a complementary approach that can be combined with promising and innovative laboratory methods such as lab-on-a-chip technologies, which allow for incorporating interactions with the tumour microenvironment. Along those lines, the integration of patient-specific information, relying on genetic and imaging data, could enable a computationally assisted and personalised treatment approach in oncology.We note that this work constitutes a proof-of-concept for the proposed (combined agent-based and Bayesian Monte Carlo) modelling approach of in vitro chemotherapeutic experimental results. Prospectively, further experimental validation based on measurements obtained from other research labs are required to solidify the biomedical suitability and clinical applicability of our in silico approach. We suggest the development of studies where computational modelling is employed to infer experimentally verifiable predictions, and the validation of these. In this way, contradictory hypotheses can be reliably discarded, and the models that have the highest explanatory power can be identified. We highlight that our data are freely accessible and invite interested researchers to conduct additional, independent validation of our results. Along those lines, the incorporation of additional model components and experimental data are necessary to realise the usage of our modelling approach in preclinical studies. Indeed, recent experimental models allow for obtaining relevant data with regard to interactions with immune cells  and the Overall, we demonstrate the applicability of ABM as a powerful complementary tool to experimental methods for probing chemotherapeutic impact. In the future, we anticipate increased uptake of in silico methods for modelling cancer growth and the impact of treatments, in particular mechanistic computational models as well as statistics based models. This is because such models have the capability to generate concrete and verifiable predictions even when using model parameters that are not directly inferred from experimental data. The mechanistic modelling approach presented in this paper, including physics-based and pharmacokinetics/pharmacodynamics models, can help inform about likely cancer evolution and prognosis.