Oesophageal adenocarcinoma is a lethal malignancy with limited treatment options. Recent studies have identified oestrogen receptors (ERs) in this cancer, which could represent a new target for therapy. In this study, we used laboratory models of oesophageal adenocarcinoma to look for the presence of variant forms of ERs. We also assessed the response to treatment with a drug that acts through these ERs. We found that variant forms of ERs do exist in this malignancy and that some of the variants appear to be important in order for the cells to respond to treatment. This could be due to interactions between different ERs, or between ERs and other molecules that are known to be important in cancer growth. Our findings are encouraging in that drugs that act through ERs might be useful for patients with oesophageal adenocarcinoma in the future.Oesophageal adenocarcinoma is a rapidly increasing problem in which treatment options are limited. Previous studies have shown that oesophageal adenocarcinoma cells and tissues express oestrogen receptors (ERs) and show growth suppression and apoptosis in response to ER modulator agents such as tamoxifen. ERs are known to be expressed in a number of isoforms that act together to regulate cell growth and cell death. In this study, we used western blotting to profile the expression of ERα and ERβ isoforms, and expression of the oncologically related molecules p53, HER2, and EGFR, in a panel of oesophageal adenocarcinoma cell lines. The cytotoxicity of tamoxifen in the cell lines was determined with Annexin V-FITC flow cytometry, and correlations between cytotoxicity and receptor expression were assessed using Spearman’s rank-order correlation. Oesophageal adenocarcinoma cell lines showed varying cytotoxicity in response to tamoxifen. The ER species ERα90, ERα50, and ERα46, as well as p53, were positively associated with a cytotoxic response. Conversely, ERα74, ERα70, and ERβ54 were associated with a lack of cytotoxic response. The ER species detected in oesophageal adenocarcinoma cells may work together to confer sensitivity to ER modulators in this disease, which could open up a new avenue for therapy in selected patients. Few malignancies carry as poor a prognosis as oesophageal adenocarcinoma. Seventy-four percent of patients die within 12 months of diagnosis ,4. Due tOesophageal adenocarcinoma demonstrates a pronounced male predilection. One hypothesis is that female sex hormones may confer a degree of protection against the disease. A recent review summarises epidemiological data that demonstrate a lower incidence of oesophageal adenocarcinoma in women with a history of exogenous oestrogen exposure  compared to those with no such history . HoweverOestrogen receptors have been detected in oesophageal adenocarcinoma biopsy tissue and resection specimens ,12,13,14Tamoxifen is an interesting drug; in that it acts as a modulator of ER signalling and not simply an antagonist of the hormone oestrogen. Tamoxifen exerts a variety of effects through the activation or inhibition of ER pathways depending on local cell or tissue context. ERs are expressed as two subtypes, ERα and ERβ, which govern many aspects of tissue proliferation and cell death . FurtherIn addition to the interaction between different isoforms, ERs also exhibit crosstalk with signalling networks that are relevant to oncogenesis, in particular, p53, EGFR, and HER2. In breast cancer, ERα binds to p53 and inhibits its activity in the DNA damage repair pathway, promoting the growth and survival of neoplastic cells and facilitating cancer progression . AdditioIn this study, we have assessed ERα and ERβ expression in a number of oesophageal adenocarcinoma cell lines, in order to ascertain which ER species are expressed, together with the expression of p53, EGFR, and HER2. We also assess the cytotoxic activity of the tamoxifen metabolite 4-hydroxytamoxifen in these cell lines. Our purpose in this is to understand whether particular ER isoforms are associated with sensitivity or resistance to tamoxifen in oesophageal adenocarcinoma, as they are in other cancers. Eight oesophageal adenocarcinoma cell lines  were used. OE19 and OE33 were purchased from Sigma ECACC , the remaining oesophageal cell lines were kindly provided by Dr Nicholas Clemons from the Sir Peter MacCallum Department of Oncology, University of Melbourne, Australia. The breast cancer cell line MCF-7 was a gift from Dr Robyn Meech from the College of Medicine and Public Health, Flinders University, Adelaide, Australia. Characteristics of cell lines are summarised in 2 and subcultured when they reached 60–80% confluence. For cell harvest, the spent medium was aspirated and cultures were washed with phosphate-buffered saline (PBS) prior to incubation with 0.5% trypsin-EDTA to facilitate cell detachment. In the semi-adherent (Eso-26) and anchorage-independent (Eso-51) cell lines, floating cells in suspension were collected prior to harvest of any adherent cells by trypsinisation. Complete medium was added in at least a two-fold excess to deactivate the trypsin and the cell suspension was centrifuged at 1500× g for 5 min. The supernatant was aspirated and cells resuspended in 10 mL of culture medium. An aliquot of the cell suspension was stained with trypan blue to identify nonviable cells and cells counted in a haemocytometer. Generally, 2–4 × 106 cells were subcultured per 75 cm2 flask. Cell culture media and reagents were purchased from Life Technologies . Because of the potential oestrogenic effects of phenol red and its possible interference with MTS assays, all culture media were phenol-red-free [Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heat-inactivated foetal bovine serum (FBS) supplemented with penicillin (100 units/mL), streptomycin (100 μg/mL), and Normocin (100 μg/mL). Cultures were maintained in a humidified environment at 37 °C with 5% COred-free .50 values calculated for three oesophageal adenocarcinoma cell lines in preliminary experiments , and within the cytotoxic range reported in the literature [For experiments, each cell line was cultured to 70% confluence, photographed, harvested, and counted. All experiments used RPMI medium supplemented with charcoal-stripped foetal bovine serum (CSS), to avoid the potentially confounding effects of steroid hormones contained within regular FBS . A conceterature ,22,23,242 flasks . Seeding densities were 4000 cells/well in 89 μL medium in 96-well plates, and 44,700 cells/well in 1 mL medium 12-well plates. Cells were plated in six technical replicates in 96-well plates for the MTS viability assay and in three technical replicates in 12-well plates for the apoptosis assay. After 24 h, drug (or vehicle control) was added to give a final drug concentration of 10 μM per well, final vehicle concentration 0.1% ethanol, and 100 μL/well in 96-well plates and 1.12 mL/well in 12-well plates. Cells were incubated for 48 h prior to harvest for viability and apoptosis assays. As with the drug concentration used, the drug exposure time of 48 h was determined by preliminary experiments assessing cell growth characteristics and different exposure times.Cells were seeded simultaneously in 96-well plates (for MTS viability assay), 12-well plates (for flow cytometry apoptosis assay), and 25 cm2 flask of each cell line was seeded in stripped medium at a density and concentration equivalent to 4000 cells/well in 96-well plates . This was harvested after 24 h, that is, at the time at which the treatments were to be added to the 12- and 96-well plates, so that a matched Western blot could be performed to assess protein expression at exactly the time of treatment. This was done to permit comparisons between receptor expression and treatment response.At the time of seeding the plates, a 25 cmAfter the treatment duration of 48 h, 20 μL of MTS reagent (333 μg/mL MTS with 25 μM phenazine methosulphate) was added to each well of the 96-well plates, and plates were incubated for 2 h. Optical density at 490 nm was measured on a Bio-Rad plate reader; mean background optical density was subtracted from the readings in each well prior to analysis.2), annexin V-FITC (0.15 mg/mL), and propidium iodide (50 μg/mL) were purchased from Abcam . Cells were harvested from 12 well plates for flow cytometry as follows. Cell medium (containing floating cells) was collected in 10 mL tubes; wells were washed with 1 mL PBS which was also collected; 500 μL trypsin was added to each well, and plates were incubated for the briefest possible time to allow for cell detachment . Cells were closely monitored during trypsinisation to ensure this step was as brief as possible in order to avoid enzymatic exteriorisation of phosphatidylserine and potential membrane injury, both of which can adversely affect the assay [g for 5 min, the supernatant was aspirated and the cell pellet resuspended in 250 μL binding buffer. Care was taken to resuspend the cells very gently, as mechanical membrane disruption can lead to exposure of phosphatidylserine to annexin V, and therefore, to false-positive results in this type of assay [Flow cytometry binding buffer . A total of 2000 events were collected per sample. Gating was applied to the forward scatter/side scatter plot to exclude cell doublets and small particles of debris. Axis scales on the FL1 (annexin V-FITC) and FL2 (PI) plots were adjusted according to thresholds determined by calibration samples for each cell line. Calibration samples comprised untreated healthy control cells with no annexin V or PI added (to identify the viable cell quadrant); treated cells with annexin V only (to determine the early apoptosis quadrant); and treated cells with PI only (to identify the late apoptosis/necrosis quadrant). In order to compare responses between different cell lines, survival fractions were calculated, and early and late apoptosis were normalised to vehicle controls according to the following formulae:Survival fraction (%) = 100 × % viability of replicate ÷ % mean viability of control replicates.Early apoptosis (%) = % early apoptosis of replicate − % mean early apoptosis of control replicates.Late apoptosis (%) = % late apoptosis of replicate − % mean late apoptosis of control replicates.One-way ANOVA followed by Tukey’s post hoc test of multiple comparisons was used to assess the significance of differences in response between cell lines. Normalised data were compared with Spearman correlation.2 flask of each cell line was also seeded for the preparation of cell lysates from untreated cells at the time of treatment. These flasks were seeded from the same cultures as those used in the treatment experiments, at exactly the same density and concentration as the treatment experiments, in order to reflect cellular protein expression at the time of treatment as precisely as possible. In addition to the oesophageal adenocarcinoma cell lysates, the breast cancer cell line MCF-7 was cultured in identical conditions and harvested for a control sample. Concurrently with treatment experiments, a 25 cmg for 5 min, and the supernatant aspirated. The cell pellet was resuspended in lysis buffer containing 1% (v/v) NP-40, 4 μM sodium azide, 50 mM Tris-HCl, and protease and phosphatase inhibitor cocktails, and sonicated at 4 °C in a water-cooled sonication unit. Lysates were centrifuged at 10,000× g for 10 min, the pellet of cellular debris discarded, and the sample stored in aliquots at −80 °C.Buffers and reagents were purchased from Bio-Rad , except where otherwise stated. Cells were harvested and lysates prepared as follows. Cells were harvested by scraping, centrifuged at 1500× The protein content of lysates was quantified using an EZQ Protein Quantification Assay  according to the manufacturer’s instruction. Briefly, lysates were spotted onto assay paper in triplicate along with a dilution series of an ovalbumin standard (from 2 mg/mL to 0.05 mg/mL), fixed with methanol, and imaged at 300 nm transillumination with a Bio-Rad Chemi-Doc MP imager. A standard curve was constructed from the relative intensity of the ovalbumin dilutions, and protein concentrations were calculated by interpolation using Carestream image analysis software .Polyacrylamide gel electrophoresis was performed as follows. Per well, 20 μg protein was made up to a volume of 20 μL per well using Laemmli sample buffer and 65 mM DTT. Samples were reduced by heating to 95 °C for 2 min and allowed to cool at room temperature prior to loading the gel. Reduced samples were loaded in pre-cast Bio-Rad Criterion TGX 4–20% stain-free gels and electrophoresis was performed at 160 V for 70 to 120 min in a gel tank filled with running buffer. Precision Plus Dual protein standards (Bio-Rad) were loaded in appropriate lanes as molecular weight markers. Recombinant ERα and ERβ were diluted 1 in 1000 and also made up in the appropriate proportions to 20 μL in Laemmli sample buffer. Following electrophoresis, gels were activated under ultraviolet light for 5 min, and imaged for 5 to 10 s (ensuring no sample lane saturation) before and after transfer to PVDF membranes, to give pre- and post-transfer images of the gels. These were used to confirm the complete transfer of protein from gel to membrane.Western blotting was performed as follows. Separated proteins were blotted onto methanol-activated 0.45 μm PVDF membranes in a Trans-Blot Turbo transfer unit (Bio-Rad) at 20 V (max 1.5 A) for 30 min. Total protein load images were obtained with a ChemiDoc MP imager (Bio-Rad) with an exposure time of 5 to 10 s, ensuring that no pixel saturation of protein bands occurred. PVDF membranes were incubated in blocking buffer for 1 h and then in primary antibody diluted to its working concentration in PVDF membrane buffer overnight at 4 °C on an agitator. Primary antibodies were HC-20 for ERα and H-150 for ERβ . Membranes were washed thrice in membrane buffer prior to incubation with a 1 in 3000 dilution of horseradish peroxidase-conjugated secondary antibody  for 1 h. Membranes were again washed thrice, and Amersham Enhanced Chemiluminescence (ECL) Western blotting detection reagent  was added immediately prior to development with a Fuji LAS-3000 Imager  running ImageLab software (Bio-Rad). Sizing and quantification analysis was performed using ImageLab software, with sizing employing a point-to-point semi-log method in which molecular weight of species of interest is interpolated from curves generated from the molecular weight markers.Quantification analysis was performed after normalisation to total protein load, as previously described . In brieRelative protein expression levels were calculated and correlated against response to tamoxifen (as measured by the flow cytometry apoptosis assay) with Spearman analysis. The expression of ER species was also compared with the expression of other ER species by Spearman correlation.Lysates from the above experiments were also profiled for p53, EGFR, and HER2 expression. Lysate preparation for Western blot, and conditions for electrophoresis, were identical to the above except for the electrophoresis duration, which was 70 min. Primary antibodies were DO-1 for p53 (Santa Cruz Biotechnologies), CST #2232 for EGFR, and CST #2242 for HER2 . Protein quantification, normalisation to total protein load, and statistical analysis were performed as described above.ERα and ERβ expression was profiled in eight oesophageal adenocarcinoma cell lines. Along with the oesophageal cell lines, control lysates from the breast cancer cell line MCF-7, and samples prepared from recombinant ERα or ERβ , were analysed. Western blots for ERα and ERβ are shown in Five major ERα species were observed, with estimated molecular weights of approximately 90 kDa, 74 kDa, 70 kDa, 50 kDa, and 46 kDa. In MCF-7, an additional species at approximately 66 kDa was observed, consistent with the reported molecular weight of wild-type ERα, and with a similar estimated molecular weight to recombinant ERα. Two ERβ species were observed, resolving at approximately 59 kDa and 54 kDa in all oesophageal adenocarcinoma lines and MCF-7. Recombinant ERβ resolved as a band with a relative molecular weight of approximately 54 kDa. Relative expression levels of ERα and ERβ species, after normalisation to total protein load, are presented in To investigate the potential relationships between ER species in oesophageal adenocarcinoma cells, relative expression was compared in a correlation matrix . The mosTamoxifen treatment at 10 μM for 48 h led to statistically significant reductions in cell viability and concomitant increases in apoptosis in all eight cell lines. OE-33, Eso-26, and Eso-51 showed the highest levels of cytotoxicity; SK-GT-4 showed the lowest; the remaining cell lines showed a spectrum of responses within an intermediate group .Since ER signalling pathways are known to interact with several pathways that are highly relevant to oncogenesis, the expression of three key molecules  was assessed in oesophageal adenocarcinoma cells. The reasons for this were (i) to examine whether there was any association between the expression of ER isoforms and the expression of these key molecules, which may imply a degree of regulatory dependence, and (ii) to assess whether there was a relationship between their expression and treatment response, since tamoxifen may interact with these pathways in certain contexts. Data represent the pooled results from three independent Western blots prepared from lysates collected during the experiments at the time treatments were applied. Representative blots are presented in p53 was detected in five cell lines, with the highest expression in Flo-1, intermediate expression in Eso-26, and lower expression in Eso-51, JH-EsoAd1, and OE-33. OACP4C, OE-19, and SK-GT-4 had no detecTable 53 kDa protein , although, interestingly, at higher exposures, OE-19 displayed a species with a molecular weight of approximately 44 kDa that reacted with the p53 antibody. EGFR was detected in all eight cell lines, with the highest relative expression in JH-EsoAd1, OE-33, and SK-GT-4. HER2 was detected at high levels in OE-19, moderate levels in Eso-26 and OE-33, and very low levels in OACP4C.TP53 missense mutation within this cell line might have a negative impact on treatment response , indicating greater sensitivity in cell lines with higher p53 expression , as shown in p = 0.0291). The difference in treatment response versus HER2 expression did not meet the threshold for statistical significance . Overall, the results suggest an association between p53 and response to tamoxifen, and between EGFR and a degree of resistance to tamoxifen.T-tests on data grouped into high vs. low expression showed that p53-positive cell lines were more sensitive to 4-hydroxytamoxifen than those in which p53 was not detected , ERα70 , and ERβ54 , as presented in = 0.7413, p = 0.0881), ERα50 , and ERβ59  were below the threshold for statistical significance.Oestrogen receptor expression data were correlated with treatment response data from the apoptosis assay by Spearman analysis. Significant correlations were found between cell survival and expression of ERα90 . Although the mutations in the cell lines examined here are predicted to produce deleterious or non-functional consequences ,29, seveTP53 that encodes a premature stop codon, resulting in a truncated p53 variant [Unlike the other cell lines, Flo-1 showed no association between p53 expression and response to tamoxifen treatment, despite its relatively high p53 expression by western blot. COSMIC and IARC databases list Flo-1 as containing p53 mutated by a missense substitution, with predicted non-functional or deleterious functional consequences. The implication is that the dysfunctional p53 expressed in Flo-1 exhibits altered responses to ER modulators, potentially precluding the study of tamoxifen metabolites in this cell line. Relative p53 expression in this study  is broadly in keeping with reported expression in these cell lines ,38, thou variant .In the analysis of ER species and response, there are two groups of ER isoforms. The first group comprises ERα90, ERα50, and ERα46, and corresponds with tamoxifen sensitivity; the second comprises ERα74, ERα70, and ERβ54, and corresponds with tamoxifen resistance. These groups are expressed in inverse proportion to each other, implying regulatory co-dependence, which is a known feature of ER isoform function . There iThe 66 kDa ERα species detected in breast cancer cell line MCF-7 corresponds in molecular weight to the recombinant protein and is likely to represent full-length ERα. This species is not detected in oesophageal adenocarcinoma cells, though there are a number of higher- and lower-molecular weight species expressed in both oesophageal and breast cancer cells. Given the proclivity of ER isoforms to co-regulate the expression of other isoforms , it is pThe 50 kDa and 46 kDa ERα species may represent the known isoforms ERα-short , ERα46 , or ERα36 . Post-translational modifications are relatively common in ER biology and can significantly alter electrophoretic mobility . Thus, tThe higher molecular weight ERα species expressed in MCF-7 and the oesophageal cell lines  are less commonly described in the breast cancer literature. An 80 kDa ERα isoform has been described in the MCF-7:2A cell line, which is a clone of MCF-7 that was cultured in oestradiol-free medium for a protracted period ,45. AnotAn alternative theory is that the higher molecular weight species may represent dimers. Dimerisation is an integral aspect of ERα and ERβ function, and dimers with resistance to typical reducing conditions have been described. Activated ER tends to dimerise in the presence of ligand, prior to translocation to the nucleus and initiation of DNA transcription at oestrogen response elements . DimerisThe two ERβ species detected by western blot at approximately 59 and 54 kDa are compatible with ERβ1 (59.2 kDa) and ERβ2 (55.5 kDa), and their negative relationship with ERα expression is in keeping with the described roles of these isoforms in the breast cancer literature . EquallyEGFR was widely expressed in oesophageal adenocarcinoma cell lines and was associated with resistance to 4-hydroxytamoxifen. EGFR overexpression is a common feature in oesophageal adenocarcinoma, with higher expression being associated with adverse clinical outcomes and resistance to chemotherapy . In breaWe acknowledge several limitations of our study. The observations reported here are associative, and causal relationships between ER isoform expression and response have not been demonstrated. Furthermore, the ERα and ERβ species reported here have not been fully characterised. Future work to characterise the ERα and ERβ species we describe, for instance using either isoform-specific antibodies or mass spectrometry, could clarify which isoforms are expressed and address the questions above around dimer formation and post-translational modification.An additional limitation is that much of the epidemiological data implicating oestrogens as a protective factor in oesophageal adenocarcinoma is derived from female patients, but the experiments presented here were performed in cell lines derived largely from males (7 male, 1 female; see A further limitation is that while the expression of related molecules p53, EGFR, and HER2 was profiled at a protein level using Western blot, an analysis of their mutation status at a genetic level was not determined. As such, the influence of mutant forms of these proteins upon cell growth and response to pharmaceutical agents in the experimental models we employed is unknown.To conclude, it is evident that tamoxifen is cytotoxic in oesophageal adenocarcinoma cells in vitro. Several ERα and ERβ species are expressed in these cell lines, and their expression is associated with either sensitivity or resistance to ER modulator therapy. This may occur due to interactions between different ER isoforms, and potentially via interactions between these isoforms and other oncologically related pathways such as p53, EGFR, and HER2. This suggests that ER modulators such as tamoxifen, acting via ERα and ERβ networks to induce a cytotoxic response, may have a role in the clinical treatment of patients with oesophageal adenocarcinoma. Further investigation to clarify the identity of the ER species associated with sensitivity or resistance to tamoxifen may help in developing a targeted approach to patient treatment.


BRAF and NRAS. Despite significant progress in therapy, targeting this signaling pathway solely has not been the solution for stopping the progression of this disease. Recently, researchers recognized the involvement of the Hedgehog-GLI (HH-GLI) signaling pathway in melanoma and its crosstalk with the MAPK pathway. In order to identify new HH-GLI-regulated targets that could be involved in the crosstalk with the MAPK pathway, we performed RNA sequencing and ChIP sequencing on three melanoma cell lines. By combining RNA-seq and ChIP-seq results, we successfully validated 15 novel targets of GLI proteins in melanoma cell lines. These findings will contribute to a better understanding of the GLI code and its role in melanoma.The majority of melanomas show hyperactivation of the MAPK signaling pathway, most often through mutations in Background: Despite significant progress in therapy, melanoma still has a rising incidence worldwide, and novel treatment strategies are needed. Recently, researchers have recognized the involvement of the Hedgehog-GLI (HH-GLI) signaling pathway in melanoma and its consistent crosstalk with the MAPK pathway. In order to further investigate the link between the two pathways and to find new target genes that could be considered for combination therapy, we set out to find transcriptional targets of all three GLI proteins in melanoma. Methods: We performed RNA sequencing on three melanoma cell lines  with overexpressed GLI1, GLI2, and GLI3 and combined them with the results of ChIP-sequencing on endogenous GLI1, GLI2, and GLI3 proteins. After combining these results, 21 targets were selected for validation by qPCR. Results: RNA-seq revealed a total of 808 differentially expressed genes (DEGs) for GLI1, 941 DEGs for GLI2, and 58 DEGs for GLI3. ChIP-seq identified 527 genes that contained GLI1 binding sites in their promoters, 1103 for GLI2 and 553 for GLI3. A total of 15 of these targets were validated in the tested cell lines, 6 of which were detected by both RNA-seq and ChIP-seq. Conclusions: Our study provides insight into the unique and overlapping transcriptional output of the GLI proteins in melanoma. We suggest that our findings could provide new potential targets to consider while designing melanoma-targeted therapy. BRAF gene, while 15–20% have a mutation in the NRAS gene [Melanoma is known as the most aggressive and deadliest of all skin cancers. The most often dysregulated signaling pathway in melanoma is RAS/RAF/MEK1/2-ERK1/2 (MAPK pathway). Almost 50% of all melanomas have a mutation in the RAS gene , making RAS gene ,7,8,9,10RAS gene ,12,13,14RAS gene . There aRAS gene . PreviouRAS gene . SurprisRAS gene . In ordeTen human melanoma cell lines  were kindly provided by Andreja Ambriović Ristov, PhD and Neda Slade, PhD. Cell lines HS895.SK , HS895.T , HS940.T  and SKMEL2  were purchased from the ATCC . HS895.SK cell line represents a healthy control: skin keratinocytes isolated from the same patient as the HS895.T melanoma cell line. All cell lines were maintained in recommended medium: Dulbecco’s Modified Eagle Medium , RPMI 1640 medium , or Eagle’s Minimum Essential Medium , supplemented with 10% FBS , 1 mM sodium pyruvate, 1% streptomycin/penicillin and 4 mM L-glutamine .GLI transfection experiments, cells were plated at density of 3 × 105 cells/well in 6-well plates, left to attach for 24 h, and then transfected with 5 μg of GLI expression plasmids: GLI1 , GLI2 and GLI3  using the X-fect reagent  following the manufacturer’s instructions.For the For determining cell viability and proliferation, compound 3--2,5-diphenyltetrazolium bromide (MTT) was used as previously described . Cells w® 20 Detergent) and incubated for 1 h with appropriate secondary HRP-conjugated antibodies, anti-rabbit 1:6000  and anti-mouse 1:8000 . Proteins were visualized using SuperWest Signal Pico and Femto reagents  on Uvitec Image Alliance 4.7 instrument .Whole-cell protein extraction, determining the protein concentration, and western blot technique were performed as previously described . The memQ61R), and A375 (BRAFV600E) were transfected with expression plasmids for GLI1, GLI2 or GLI3 in two independent experiments. Non-transfected lines were used as controls. Briefly, 200,000 cells were seeded in a Ø10 cm cell culture dish and transfected the next day with 5 µg of plasmid DNA, using the X-fect reagent  and following the manufacturer’s instructions. Forty-eight hours post-transfection, total RNA was isolated with Absolutely RNA miRNA Kit . The quality of the isolated RNA was checked on agarose gel, and RNA concentrations and purity were measured on NanoPhotometer N60 instrument . Sequencing libraries were generated and sequenced by DNA Link Company . The integrity of RNA samples was checked on Agilent 2100 Bioanalyzer . The instrument provided data on RNA concentration, electropherogram, rRNA subunit ratios, and RNA integrity number (RIN). All samples had RIN ≥ 8.0 and a 28S:18S ratio ≥ 1.4. Total amount of 1 µg of RNA was used. cDNA libraries were prepared using TruSeq mRNA library Kit, and sequencing was performed on Novaseq 6000 instrument . Human melanoma cell lines CHL-1 (wild-type for both NRAS and BRAF), MEL224  glutarate (DSG)  followed by 1% formaldehyde. Samples were incubated with specific ChIP-grade antibodies against GLI1 10 μg/sample  [Chromatin immunoprecipitation was performed using SimpleChIP Plus Enzymatic Chromatin IP kit  following the manufacturer’s protocol. Three cell lines with the strongest basal expression of GLI proteins were selected for the experiment . Cells were cultured until 80–90% confluence in 150 cmhttps://github.com/NenBarto/GLI (accessed on 10 August 2022).FastQC software (v.0.11.5) was used to assess the sequencing quality of the raw fastq data. The sequencing reads have been trimmed from adapters with Trim galore (v.0.3.7) and then mapped to human genome hg38 with STAR aligner (v.2.4.0d) with the following parameters: sjdbOverhang 99, outFilterMismatchNoverReadLmax 0.04, outFilterMultimapNmax 500, outSAMmultNmax 1. Mapped reads were then quantified with RSEM (v.1.2.26) over hg38 gencode transcriptome (v.28). Further analysis was performed with R (v.4.0.1), and differential gene expression was performed with edgeR (v.3.30.3) for each cell line with overexpressed GLI against the control. Gene Set Enrichment Analysis of KEGG pathways was performed with function gseKEGG from R package clusterProfiler (v.3.0.4). Data were visualized in R with custom scripts, available at https://github.com/NenBarto/GLI (accessed on 10 August 2022). Motif enrichment was performed with MEME Suite v.5.3.0 [ChIP-seq libraries were first trimmed with trimgalore from FastQC (v.0.11.5) and mapped with BWA v0.7.9a to human genome hg38. After removing duplicates with picard-tools (v. 1.138), peaks were called with MACS (v.2.0.10) with parameters -f BAMPE -g -B -q 0.01. Further analysis was performed in R (v.4.0.1) and packages ChIPpeakAnno and ChIPseeker using custom scripts available at  v.5.3.0 . The lis v.5.3.0  and iden v.5.3.0  to examiRPLP0 and TBP housekeeping genes. Primer sequences used for qPCR are listed in Total RNA was extracted using TRIzol reagent  following the manufacturer’s instructions. cDNA was generated from 1 μg of RNA using the High-Capacity cDNA synthesis kit  and qRT-PCR performed on CFX-96 instrument  using SsoAdvanced SYBR Green Supermix  with gene-specific primers. Fold change was calculated relative to the p-values < 0.05 were considered statistically significant.D’Agostino–Pearson test was used for testing normality of data distribution. Non-normal data were log-transformed. An independent samples T-test or One-way ANOVA with Dunnett’s post hoc test was used for inferring the differences in gene expression. Statistical analyses were performed with MedCalc v19.2.1 . Two-tail GLI1, GLI2, GLI3, and PTCH1) on a panel of 14 human melanoma cell lines with different genetic backgrounds. Five cell lines  are wild-type for both BRAF and NRAS gene; five cell lines  have a BRAFV600E mutation; three cell lines  have an NRASQ61R mutation, and the MEL505 cell line has a KRASG12V mutation. BRAF or NRAS mutation status is not correlated with the differences in protein or gene expression between the cell lines, but KRAS mutated MEL505 cell line shows lower gene expression levels for all tested genes , which was previously reported for GLI2 [EBI3, shows a broad peak in the promoter region of the gene  were selected. ChIP-seq datasets were merged across cell lines to observe overall GLI binding sites and increase the signal-to-noise ratio. More than 80% of ChIP-seq peaks were identified in the intergenic regions A,B, whicfor GLI2 , and itsthe gene E.V600E homozygous), SKMEL24 (BRAFV600E heterozygous), MEL224 (NRASQ61R homozygous), SKMEL2 (NRASQ61R heterozygous), MEL505 (KRASG12V heterozygous), CHL-1 and MEWO (wild-type for BRAF and NRAS). Selected cell lines were transfected with GLI1, GLI2, or GLI3 expression plasmids, and gene expression of a total of 23 genes  was determined. To narrow down a list of targets for qPCR validation, two approaches were used , confirming the validity of the selection process. A comparison of GLI3 RNA-seq and ChIP-seq data showed no common targets. The chIP-seq analysis identified GLI1 or GLI2 binding motifs in all 11 identified targets, suggesting they are direct transcriptional targets of GLI1 and/or GLI2 proteins. The second approach was to analyze the DEGs from RNA-seq independently of the ChIP-seq data to identify potential indirect targets. After filtering by FDR and logFC values, the GeneAnalytics tool of the GeneCards database  was used to identify the role of DEGs in signaling pathways and diseases. Several categories of pathways and diseases with a high relevance score were considered when selecting genes of interest: “Pathways in cancer”, “PI3K-AKT signaling pathway”, “MAPK signaling pathway”, “WNT/Hedgehog/NOTCH”, “neoplasm”, “melanoma” and “abnormalities of the skin”, as well as logFC values of these genes in RNA-seq analysis. Additional screening was performed based on their expression in melanoma from the GEPIA database [KRT16, KRT17, S100A7, S100A9, GH1, SOX9, BIRC7, MRAS, RET, and IL1R2. Finally, 21 targets were selected for qPCR validation: 10 identified by RNA-seq only  and 11 targets identified with both ChIP-seq and RNA-seq  —RAB34, LAPTM5, RDH10, and STK32C—exhibited a consistent and uniform expression pattern. In addition, the genes EBI3, GH1, SOX9, RET, and SPRY2 are also good candidates for HH-GLI targets but exhibited a less uniform expression pattern in these melanoma cell lines. Overall, by combining RNA-seq and ChIP-seq results and elaborate filtering of these genes, we successfully validated 15 novel targets of GLI proteins in melanoma cell lines.To validate the biological reproducibility of the results of DEG analysis, we performed qPCR experiments on seven melanoma cell lines. Cell lines were chosen to represent the different mutational backgrounds: A375 (BRAFere used A. The fidatabase  (SKCM dand EBI3) B,C. A suBRAF mutation are more sensitive to SMO inhibitor, sonidegib, than BRAF wild-type cells [V600E mutated cell lines seem to be more sensitive to inhibitor GANT61 than cell lines that are wild-type for these genes, but in our case, this difference is not statistically significant. Thus far, studies have demonstrated that in colon cancer, neuroblastoma, and pancreatic cancer, GANT61 is the most effective inhibitor of cell growth among all tested HH-GLI inhibitors [BRAF inhibitors have improved patient survival compared with standard chemotherapy, but these benefits are not persistent, as most patients develop resistance to therapy which leads to disease progression . There ape cells . We alsohibitors ,113,114.hibitors . Our MTThibitors ,104,107.BRAF mutation, an NRAS mutation, or no mutation in these two genes. We applied RNA sequencing and combined it with ChIP sequencing to identify direct but also unique and overlapping targets of GLI1, GLI2, and GLI3 in three melanoma cell lines. By doing so, we identified a total of 808 DEGs for GLI1, 941 DEGs for GLI2, and 58 DEGs for GLI3. KEGG analysis confirmed that many of the identified DEGs are involved in various signaling pathways, including MAPK, Ras, Hippo, and Wnt pathways [KRT16, KRT17, S100A7, MRAS, BIRC7, IL1R2, as well as several direct GLI targets (confirmed by both RNA-seq and ChIP-seq)—RAB34, LAPTM5, RDH10, and STK32C—have a consistent and uniform expression pattern. Expression levels of these genes are increased with GLI1 or GLI2 overexpression, regardless of the mutational status of the cell lines. We were also able to validate genes EBI3, GH1, SOX9, RET, and SPRY2 as HH-GLI targets. Their expression was consistent in the majority of the cell lines, with few exceptions. Six targets—S100A9, FLG, SAMMSON, LY6D, CACNA2D2, and HES1—were found to have variable expression in different melanoma cell lines, so they could not be validated as GLI targets in melanoma. Because the exact interplay between the HH-GLI pathway and MAPK signaling pathway is not yet understood, we decided to investigate the transcriptional targets of all three GLI proteins in melanoma with different genetic backgrounds, either harboring a pathways , as wellpathways ,118,119.KRT16, S100A9, SOX9, BIRC7, EBI3, FLG, SAMMSON and SPRY2—were already implicated in melanoma pathogenesis [KRT17 closely follow those of KRT16 in all seven melanoma cell lines . In the majority of our melanoma cell lines, S100A9 expression could not be detected. By contrast, S100A7 shows a much wider expression pattern than S100A9, but despite its ability to promote cell proliferation, migration, invasion, and tumor metastasis in cervical, breast, and ovarian cancer [MRAS, IL1R2, RAB34, LAPTM5, RDH10, and STK32C. Similar to S100A7, we considered them important because of their involvement in processes of tumor cell proliferation, migration, and invasion, or their interactions with members of the MAPK cascade. For example, it is shown that cells overexpressing MRAS have higher migratory potential and that MRAS/SHOC2/SCRIB complex coordinates ERK pathway dynamics [STK32C inhibited tumor cell proliferation, migration, and invasion in human bladder cancer cells [hibitors  and in mhibitors . Our resn cancer ,43,44,45dynamics . It has dynamics . One studynamics . It has dynamics  and thater cells . Finallyer cells .FLG as the overlapping target of GLI1, GLI2, and GLI3 and detected it with both ChIP-seq and RNA-seq, its expression levels detected by qPCR experiments are not consistent between the cell lines. A375 cell line shows increased expression levels of FLG in all GLI overexpressed samples, while SKMEL24 shows a decrease in FLG expression levels. In the other cell lines, FLG expression could not be detected with qPCR. A previous study has noted the important role of the long noncoding RNA (lncRNA) SAMMSON in melanoma [SAMMSON has been shown to be important for human melanoma cell growth and survival while also highlighting the role of a SAMMSON in modulating the adaptive resistance of mutant BRAF melanoma to RAF inhibitors [SAMMSON, SPRY2 has also been implicated in resistance to BRAF inhibitors [SAMMSON and SPRY2 are downregulated targets of GLI1, and ChIP-seq confirms that SAMMSON has a GLI1 binding motif, while SPRY2 contains GLI1 and GLI2 binding motifs. Because GLI1 is a transcriptional activator, it is not clear how it downregulates the expression of these two targets. It is likely that some other factors, apart from GLI1, play a role in their regulation. qPCR results show that SAMMSON expression levels vary among the cell lines. For example, A375 and SKMEL2 (with GLI1 or GLI2 overexpression) exhibit decreased expression levels of SAMMSON, while other cell lines, such as SKMEL24, MEL224, and MEL505, generally show increased SAMMSON expression levels. qPCR results show that SPRY2 is detected in all melanoma cell lines, especially those with GLI3 overexpression.Although we identified melanoma . More rehibitors . Similarhibitors ,120. OurOur studies confirm that in melanoma, the HH-GLI signaling pathway is in crosstalk with other signaling pathways and that its activation is more likely non-canonical than canonical. Out of 21 selected targets, we validated 15 as novel targets of GLI proteins, considering their expression in melanoma cell lines and possession of GLI binding motifs. Our study provides insight into the unique and overlapping transcriptional output of the GLI proteins in melanoma, which will contribute to a better understanding of the GLI code and its role in tumorigenesis. Other potential targets can also be functionally validated using this data in the future, especially by researchers in the HH-GLI field that are interested in other aspects of HH-GLI signaling. Our findings provide new potential targets to consider while designing melanoma-targeted therapy, especially in the case of recurrent disease due to therapy resistance.


Notably, 3‐HAA inhibits the proinflammatory factors TNF and IL‐6 in multiple cell subsets, including resident macrophages, proliferating macrophages, and pDCs. This study reveals the landscape of immune cell subsets in HCC in response to 3‐HAA, indicating that 3‐HAA may be a promising therapeutic target for HCC.Kynurenine derivative 3‐hydroxyanthranilic acid (3‐HAA) is known to regulate the immune system and exhibit anti‐inflammatory activity by inhibiting T‐cell cytokine secretion and influencing macrophage activity. However, the definite role of 3‐HAA in the immunomodulation of hepatocellular carcinoma (HCC) is largely unexplored. An orthotopic HCC model and treated with 3‐HAA by intraperitoneal injection is developed. Furthermore, cytometry by time‐of‐flight (CyTOF) and single‐cell RNA sequencing (scRNA‐seq) analyses are carried out to define the immune landscape of HCC. It is found that 3‐HAA treatment can significantly suppress tumor growth in the HCC model and alter the level of various cytokines in plasma. CyTOF data shows that 3‐HAA significantly increases the percentage of F4/80 Multiple‐cell subsets, such as resident macrophages, proliferating macrophages, and pDCs, display significant inhibition of the proinflammatory factors TNF and IL‐6 by 3‐HAA. CXCL13 is enriched in kupffer cells and upregulated in the 3‐HAA‐treated group. 3‐HAA inhibits CSF1 and CSF2 in M2 macrophages. 3‐HAA may be involved in remodeling the tumor immune microenvironment and provides novel treatment strategies for HCC. Infection with the hepatitis B and C viruses is a prominent risk factor for the onset and progression of HCC. Although various treatment options based on liver resection, locoregional therapies, targeted therapy, chemotherapy, and liver transplantation are available, many questions remain unanswered. Only liver transplantation is considered an ideal cure, which is constrained by the availability of donor grafts. Considering the aforementioned factors, advancements in HCC treatment are still required.Primary liver cancer was the sixth most prevalent cancer and the fourth leading cause of cancer‐related mortality globally.1 Hepa Disorders of Trp metabolism can lead to apoptosis and dysfunction of immune cells, which is conducive to the formation of an immunosuppressive microenvironment, thereby affecting the efficacy of immunotherapy for HCC.Tryptophan (Trp) is an essential amino acid in humans that is involved in protein synthesis and serves as a precursor for the synthesis of many microorganisms and host metabolites. Notably, Trp is an important energy source for the regulation of immune cell function.5 Diso Kyn is converted into 3‐hydroxyanthranilic acid (3‐HAA), 3‐hydroxykynurenine, and other metabolites via kynurenine 3‐monooxygenase, kynurenine aminotransferases, and kynureninase. Studies have reported that the Kyn derivative 3‐HAA suppresses tumor growth and sensitizes HCC to sorafenib. In addition, 3‐HAA is known to regulate the immune system and exhibits anti‐inflammatory activity by inhibiting T‐cell cytokine secretion and influencing macrophage activity. However, the precise role of 3‐HAA in cancer immunity is largely unknown.There are three pathways involved in Trp metabolism, with the kynurenine (Kyn) metabolism pathway accounting for 95% of Trp metabolism. Kyn is the primary product of the Trp metabolism pathway, which is catalyzed by tryptophan 2,3‐dioxygenase and indoleamine 2,3‐dioxygenase.6 Kyn hiCX3CR1loKi67loMHCIIhi macrophages and decreased the percentage of F4/80loCD64+PD‐L1lo macrophages, but also it regulated the function of M1 macrophages, M2 macrophages, and proliferating macrophages. Our findings can precisely define the immune cell subsets that are relevant to HCC, and 3‐HAA treatment could be a promising treatment strategy for HCC.This study aimed to elucidate the effects and immunoregulation of 3‐HAA on HCC. The levels of Kyn derivative 3‐HAA in HCC tissues and adjacent nontumor tissues were determined using liquid chromatography–mass spectrometry (LC–MS). We demonstrated that 3‐HAA treatment can significantly suppress tumor growth in the HCC model. Furthermore, cytometry by time‐of‐flight (CyTOF) and single‐cell RNA sequencing (scRNA‐seq) analyses were carried out to define the immune cell landscape of HCC. Not only did 3‐HAA increase the frequency of F4/8022.1Figure  Here, we performed LC–MS to explore the different metabolites between 15 pairs of tumor tissues and adjacent nontumor tissues of HCC patients. It revealed that the level of the Kyn derivative 3‐HAA was lower in tumor tissues than in adjacent nontumor tissues, and the time‐dependent receiver operating characteristic (ROC) analyses for the survival prediction of 3‐HAA yielded the area under the curve of 0.951  compared to the control group (HCC group) Figure . To furte Figure . The res2.3Figure+ T cells, CD4+ T cells, B cells, dendritic cells (DCs), natural killer (NK) cells, neutrophils, plasmacytoid dendritic cells (pDCs), gamma‐delta T (γδT) cells, macrophages, and eosinophils ; and CD45+F4/80−CD64+CD39+CD44+CD11b+CD11c+ macrophages (C12)  Figure 4A. We pes Figure . Combine2.5Figure To further understand the tumoral immune cell remodeling mediated by 3‐HAA, we collected fresh tumor tissues from 3‐HAA group and HCC group for scRNA‐seq with the 10x drop‐seq platform (10x genomics). 2.6Figure CXCL2 and CCL5), antioxidant gene (Hmox1), and immunoregulatory genes (Chil3) were upregulated in M1 macrophages  was found to be upregulated after 3‐HCC treatment. Notably, 3‐HAA treatment significantly upregulated the expression levels of Ly6C in macrophage and downregulated the expression level of chemotactic CCL2 produced by macrophages in HCC tissues, suggesting that 3‐HAA may inhibit HCC progression by remodeling the immunophenotyping of macrophage by inhibiting the CCL2/CCR2 pathway.The phenotypic switching of Ly6C disease.24 NumConclusion, our study reveals the influence and impact of 3‐HAA in regulation immune microenvironment in HCC and initially suggests 3‐HAA as a novel strategy of therapy for restoring immune function in HCC. However, the molecular mechanism of 3‐HAA in immunoregulation and the clinical data to support the therapeutic effect of 3‐HAA need further study.4n = 15) from HCC patients were obtained during surgical resection. Once the tissues were obtained during surgery, they were quickly frozen in liquid nitrogen and stored at −80 °C for further analysis.The study was approved by The First Affiliated Hospital, Zhejiang University School of Medicine, and all participants signed written informed consent (project number IIT20210168B‐R1). Tumor tissues and adjacent nontumor tissues  solution in 50% acetonitrile and filtering the supernatant through a 0.22 µm membrane. Chromatographic separation was carried out in a Thermo Scientific Vanquish system equipped with Waters Corp ACQUITY UPLC HSS T3  column kept at 40 °C. The ESI‐MSn experiments were carried out on the Thermo Scientific Q Exactive HF‐X mass spectrometer with a spray voltage of 3.5 kV and −2.5 kV in positive and negative modes.LC–MS was performed on 15 HCC tissues and 15 adjacent nontumor tissues from 15 HCC patients, as previously described.29 In 2 at 37 °C, as previously described. Six‐week WT C57BL/6J male mice were anesthetized with 1% pentobarbital solution  at a dose of 50 mg kg−1. The mice were secured in the supine position on operating table, and their abdomen was sterilized. Sterilized tweezers and ophthalmic scissors were used to open the mice abdominal cavity. This procedure was carefully performed to avoid any cuts in the blood vessels and unnecessary bleeding. The left lobe of the liver was plucked out using a sterile cotton swab. 3 × 105 Hepa1‐6 cells suspended in 10 µl Corning Matrigel (Matrigel:PBS = 1:4) were injected into the left lobe of the liver. Following the application of sterile cotton swabs to stop the bleeding, the mouse's abdomen was sutured. Finally, mice naturally woke up in the incubator. 3‐HAA was purchased from Aladdin Bio‐Chem Technology Co., Ltd.  and administered intraperitoneal injection at a dose of 100 mg/kg once daily for consecutive 14 days. This experiment was approved by the Animal Experimental Ethics Committee of The First Affiliated Hospital, Zhejiang University School of Medicine (project number 20221072).The liver tumor cell line Hepa1‐6 was obtained from ATCC and maintained in Dulbecco's Modified Eagle's Medium  supplemented with 10% fetal bovine serum  in a humidified atmosphere containing 5% COescribed.31 Sixn = 10 vs n = 10) were tested using Quantibody Mouse Inflammation Array Kit  according to the manufacturer's instructions. The information regarding these 40 cytokines is presented in Table  The concentrations of various cytokines were analyzed using the Student's t‐test.A total of 40 cytokines in mouse plasma samples  removed surgically from mice models were stored and transported at a low temperature. Tissues were digested using the Tumor Dissociation Kit , and then cells were filtered, washed, and stained. A total of 42 metal‐isotope‐tagged antibodies were used in the study. A final concentration of 0.25 µM 194Pt (1 mM) dead and live staining solution were prepared. Cells were resuspended in 100 µL of 194Pt dead and live staining solution and stained on ice for 5 min (100 µL per sample). 1 mL of FACS buffer  was added to each sample and centrifuged at 400 g at 4 °C for 5 min. After discarding the supernatant, 50 µL of Block mix was added to each sample, the cells were resuspended, and the samples were blocked on ice for 20 min. Following incubation, the antibody mix was added directly. The cells were stained for 30 min on ice. The Fix and Perm Buffers were used to prepare a staining solution with a final concentration of 250 n. 200 µL of resuspended cells were taken from each sample and stained for 1 h at 24 °C. All CyTOF data were normalized and manually gated in FlowJo (version 10). Circle the single and intact live cells, and label the CD45+ immune cells. The x‐shift algorithm was used for cell subgroup clustering, annotation, t‐SNE dimensionality reduction visualization, and statistical analysis.Six fresh HCC tissues  infiltrating immune cells were incubated with anti‐mouse antibodies CD45‐FITC, LIN‐, F4/80‐BV421, CD11c‐PE‐CY7, CD39‐BV711, L/D‐7AAD . All the antibodies were purchased from BD Biosciences. A Fortessa cell analyzer  was used to test stained cells.Six disaggregated mice model tumor tissues  from mice model of HCC. First, the tissues were processed into single‐cell suspensions for cell counting and cell viability evaluation. The prepared single‐cell suspension was combined with gel beads containing barcode information and a mixture of enzymes. Next, cell lysis and reverse transcription reactions were carried out in the Gel Beads‐in emulsions (GEMs). The 10x Genomics barcodes were linked to the cDNA products in the corresponding cells during this process. The GEMs were then disrupted, and the cDNAs from different cells were mixed. Based on this, PCR amplification and quality inspection (amplified fragment size and yield of amplified product) were performed on the 50 000‐100 000 reads measured per cell using the Illumina Hiseq PE150 sequencing strategy .scRNA‐seq was performed on tumor tissues . P < 0.05 (two‐sided) was considered statistically significant.The statistical analyses were carried out using SPSS 25.0 and R 3.6.1 software. Statistical differences of a continuous variable between the two groups were analyzed by unpaired Student's The authors declare no conflict of interest.C.X., X.G., Q.Z., and Q.S. contributed equally to this work. L.L. and J.L. designed and guided the study. C.X., X.G., Q.Z., and Q.S. participated in the overall experiments and wrote the draft. X.Y., Q.C., J.J., Y.S., and Z.B. collected and analyzed data. All authors read and approved the final manuscript.Supporting informationClick here for additional data file.


Their structures including absolute configurations were assigned by spectroscopic analysis, in combination with GIAO NMR shift calculation and DP4+ analysis. Structures of neo-debromoaplysiatoxin I and neo-debromoaplysiatoxin J contained a decahydro-5H-pyrano  chromen-5-one 6/6/6 ring skeleton and an intriguing peroxide bridge group, respectively, which are unprecedented structure scaffold and motif in aplysiatoxins. Two compounds displayed comparable inhibitory activities against Kv1.5 K+ channel with IC50 values of 2.59 ± 0.37 μM (1) and 1.64 ± 0.15 μM (2); however, they presented differential cytotoxic effects. It is worth noting that neo-debromoaplysiatoxin J, containing a peroxide bridge, showed remarkable cytotoxicity against four cancer cell lines including SW480, SGC7901, LoVo and PC-9 compared to the human normal cell line.Two aplysiatoxin derivatives, neo-debromoaplysiatoxin I ( The NMR data of 2 exhibited a keto carbon (δC 207.2), two carbonyl carbons (δC 169.8 and 165.5), six aromatic carbons , two oxygenated quaternary carbons (δC 105.7 and 87.2) and a methoxy (δC 56.7)  + .Neo-debromoaplysiatoxin J (2): white solid; max (log ε) 196 (3.34) nm, 275 (2.56) nm; IR (KBr) νmax: 3418 (br), 2966, 2929, 1727, 1653, 1599, 1455, 1300, 1223, 1117, 1067, 1047, 877 cm−1; 1H NMR  and 13C NMR  data see m/z 629.2938 [M + Na]+ .The calculations, conformational search and optimization were performed as reported . The sta2. When the cells grew to 70–85% confluence, they were passaged for subsequent tests. In all the tests, control cells were treated with DMSO .The mouse connective tissue fibrocytes (LTK cells) stably expressing human Kv1.5 channels (LTK-Kv1.5) from Professor Weiping Wang at Chinese Academy of Medical Sciences and Peking Union Medical College, China, was cultured for ion channel inhibitory experiment. The 293A human kidney cell line, SW480 human colorectal cancer cell line, SGC7901 human gastric cancer cell line, LoVo human colorectal carcinoma cell line and PC-9 non-small-cell lung cancer cell line, used for cell viability assay, were kindly provided by Professor Yigang Wang and Professor Wenbin Ou at Zhejiang Sci-Tech University, China. Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) , except PC-9 in RPMI-1640 medium , containing 10% fetal bovine serum (FBS)  and 1% antibiotics   at 37 °C in a humidified atmosphere containing 5% CO2 (1 mM), HEPES (10 mM), Mg-ATP (3 mM) and EGTA (10 mM). The pH was adjusted to 7.25 by KOH. The extracellular fluid used to record currents contained NaCl (135 mM), KCl (5.4 mM), MgCl2 (1 mM), CaCl2 (1.8 mM), Glucose (10 mM), HEPES (10 mM) and NaH2PO4 (0.33 mM). pH was adjusted to 7.4 by NaOH.The instruments and methods for the experiment mainly refer to previously reported . Whole-op < 0.05 was considered to be significant.Kv1.5 currents were evoked by a 300 ms depolarizing pulse from −50 mV to 50 mv in 10 mV increments from a holding potential of −70 mV. The current amplitude at the end of 300 ms pulse at 50 mV was measured. All values were indicated as mean ± SEM, and a value of 50) was calculated using GraphPad Prism (Version 8.0.2) software.The cells were seeded on 96-well plates  at a density of 3000 cells in 100 μL medium per well. After 16–20 h, the medium was changed into a fresh medium that had different concentrations of compounds. After a 72 h incubation period, 100 μL fresh medium with 10% CCK-8 reagent  was added along the hole-wall into each hole to replace the drug-containing medium. The plates were incubated for 1–2 h before the light absorption values at 450 nm of each group were detected. Cisplatin (5 μM) , a kind of broad-spectrum anticancer drug for clinical application, was used as positive control in cell viability assay. Furthermore, the half inhibitory concentration (IC1), featuring a new skeleton, and neo-debromoaplysiatoxin J (2), with an unprecedented peroxide bridge group, were characterized from cyanobacterium Lyngbya sp. Two compounds all exhibited significant inhibitory activities against the Kv1.5 K+ channel with IC50 values of 2.59 ± 0.37 μM (1) and 1.64 ± 0.15 μM (2), respectively. Surprisingly, in the in vitro cytotoxic assays, 2 exhibited apparent stronger cytotoxic effects against four cancer cell lines, including SW480, SGC7901, LoVo and PC-9, than 1, with cell viability less than 20% at 20 μM, while it exhibited no or less cytotoxicity against 293A human kidney cells  from 1 μM to 20 μM.In conclusion, neo-debromoaplysiatoxin I (


For pre-clinical drug development and precision oncology research, robust cancer cell models are essential. Patient-derived models in low passages retain more genetic and phenotypic characteristics of their original tumors than conventional cancer cell lines. Subentity, individual genetics, and heterogeneity greatly influence drug sensitivity and clinical outcome.in vitro drug sensitivity towards standard-of-care chemotherapeutic regimens was evaluated.Here, we report on the establishment and characterization of three patient-derived cell lines (PDCs) of different subentities of non-small cell lung cancer (NSCLC): adeno-, squamous cell, and pleomorphic carcinoma. The in-depth characterization of our PDCs included phenotype, proliferation, surface protein expression, invasion, and migration behavior as well as whole-exome and RNA sequencing. Additionally, The pathological and molecular properties of the patients’ tumors were preserved in the PDC models HROLu22, HROLu55, and HROBML01. All cell lines expressed HLA I, while none were positive for HLA II. The epithelial cell marker CD326 and the lung tumor markers CCDC59, LYPD3, and DSG3 were also detected. The most frequently mutated genes included TP53, MXRA5, MUC16, and MUC19. Among the most overexpressed genes in tumor cells compared to normal tissue were the transcription factors HOXB9, SIM2, ZIC5, SP8, TFAP2A, FOXE1, HOXB13, and SALL4; the cancer testis antigen CT83; and the cytokine IL23A. The most downregulated genes on the RNA level encode the long non-coding RNA LANCL1-AS1, LINC00670, BANCR, and LOC100652999; the regulator of angiogenesis ANGPT4; the signaling molecules PLA2G1B and RS1; and the immune modulator SFTPD. Furthermore, neither pre-existing therapy resistances nor drug antagonistic effects could be observed.In summary, we successfully established three novel NSCLC PDC models from an adeno-, a squamous cell, and a pleomorphic carcinoma. Of note, NSCLC cell models of the pleomorphic subentity are very rare. The detailed characterization including molecular, morphological, and drug-sensitivity profiling makes these models valuable pre-clinical tools for drug development applications and research on precision cancer therapy. The pleomorphic model additionally enables research on a functional and cell-based level of this rare NCSLC subentity. The estimated number of new cases in 2022 in the US is 236,740, and the estimate for deaths is 130,180 accordingly. Thus, lung tumors account for 12% of all new cancer cases and 21% of all cancer deaths. The 5-year survival rate was 22.9% in the years 2012–2018. However, this large tumor entity is composed of a variety of subentities. In 2021, the updated “WHO Classification of Lung Tumors” was published  types: squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Pleomorphic carcinomas in contrast account for less than 1% of lung tumors . PublishThe goal of precision oncology is to offer highly effective treatment by an individualized therapy approach subsequent to comprehensive molecular, cellular, and functional analyses of the tumors. This approach is rapidly developing and has, especially for the molecular assessments, entered the mainstream of clinical practice. Functional analyses, however, require vital cells or better patient tumor models . Thus, iin vivo models followed suit. Engrafting tumor pieces to generate PDX models is a rather recent development -throughput screening without elaborate equipment is barely feasible, and, last but not least, time and cost efficiency of adherent cell lines surpass organoids by far. Also, adherent cell lines, especially PDCs, possess a high level of predictability and utility in pre-clinical tumor therapy assessments . Thus, w22.1Cell lines were established as previously published . BrieflyThe names of the cell lines consist of the following information: pseudonymized patient ID containing information on place of material collection (HRO = Hanse City of Rostock) and tumor entity/organ of origin . Passage numbers are given for all experiments.All processes involving patients and patient-derived material were approved by the ethics committee of the University Medical Center Rostock (UMR): A 2019-0187. General guidelines for working with patient material were followed; this included obtaining written consent for each patient in advance.2.22 incubator at 37°C. All cell culture media and reagents were purchased from PAN , and all culture plates and flasks were from Sarstedt .Cells were cultured in standard cell culture flasks using DMEM/F12 medium supplemented with 10% fetal calf serum (FCS) and 2 mM L-glutamine in a humidified CO2.32.3.1The absence of contaminating mycoplasma was checked on a routine basis using cell culture supernatant and the PlasmoTest™ - Mycoplasma Detection Kit  according to the manufacturer’s recommendation.2.3.2Concordance of PDCs and patient donor tissue was confirmed by short tandem repeat (STR) analysis as previously described . In shor2.44 cells per well) were seeded in a 24-well plate and incubated for 24 h to allow attachment. One column of the 24-well plate was washed with PBS and stained with crystal violet solution every 24 h for 7 consecutive days resulting in quadruplicates for each time point. On the last day, plates were washed three times with PBS and left to dry at room temperature. After complete drying, 100 µl/well 1% sodium dodecyl sulfate solution was added, and plates were placed on a shaker for 10 min to dissolve the crystal violet. Absorbance measurements at 590 nm were performed using a Tecan Infinite 200 Pro  plate reader. Measurements were normalized to the first time point measurement, and doubling time was calculated with GraphPad Prism 9.4.1 using the exponential growth equation for nonlinear regression.Cells (2–5 × 102.55 cells/100 µl. The antibodies (1 µg per antibody and tube containing 100 µl of cell suspension) were added and incubated at 4°C for 30 min in the dark  in 150 µl/well standard medium and incubated for 24 h to facilitate attachment. The chemotherapeutics were added to the wells in 50 µl of standard medium yielding the desired final concentrations. For every single agent tested, detailed information can be found in Cells were seeded on a 96-well plate  and one row was used as living control (≙ untreated cells), leaving two 6 × 6 dose response matrices for drug sensitivity testing. We tested drug combinations that are typically used in clinical practice, by combining a platinum-based drug (cisplatin and carboplatin) with etoposide, vinorelbine, and paclitaxel, resulting in six paired combinations .2.9After preparing a single-cell suspension, cells were seeded at 1,000 cells/well in duplicate in a cell-repellant six-well plate  in 5 ml of spheroid medium  coated with 0.4% base agar and 0.35% top agar. Formation of spheres was checked daily and the final outcome (outgrowth or no outgrowth) was scored as positive or negative at day 14.2.103 cells/µl was prepared in FCS-free standard medium. A 24-well plate with TC inserts (Greiner) was prepared and 500 µl of the cell suspension (≙ 5 × 105 cells) was added to each insert. For the invasion assay, inserts were coated with Matrigel® Basement Membrane Matrix  prior to the addition of the cell suspension. Then, 750 µl of 10% FCS containing medium was added to the lower wells. After incubation for 72 h at 37°C and 5% CO2, the medium was removed, and inserts were washed twice with PBS. Finally, 1 ml of 0.2% crystal violet solution was added to each insert for cell staining. After 15 min of incubation, removal of the staining solution, and three washing steps with PBS, the non-invasive cells on the upper side of the insert were scraped off with a cotton swab. The addition of 0.75 ml of 1% SDS solution and 15 min incubation on a shaker led to solvation of crystal violet. The inserts were removed, the plate was placed in the Tecan reader, and absorbance was measured . Measurements were normalized to the HROC24 cell line . Successful DNA isolation was confirmed by measuring DNA concentration with Nano-Drop .DNA from cell pellets was extracted using the Promega Wizard2.12.2p-value threshold for calling variants was set to 0.99 and the p-value threshold for calling somatic variants was set to 0.05. The resulting variants were then filtered using vcflib . Bioinformatics were partly done on the Galaxy Europe platform , 20. Aftg vcflib  and bcftg vcflib  to retai1 Mutec2  was used1 Mutec2  database1 Mutec2  and anno1 Mutec2  functionFor mutational signature analysis, we used the maftools  R packagFor the oncoplot visualization, in order to pick 30 mutations that could be relevant, we first included genes where multiple samples have somatic mutations. All remaining mutations from the matched tumor–normal paired samples were annotated with the Catalogue of Somatic Mutation In Cancer (COSMIC) gene  databaseTumor mutational burden (TMB) calculation was done using maftools by dividing the total number of non-synonymous coding somatic mutations by the size of the human exome [30 MB ].2.132.13.1Total RNA from cell lines was isolated by using the EurX RNA Purification Kit. Total RNA from tissue was extracted using the Precellys Tissue RNA Kit (PeqLab by VWR).2.13.2p-value< 0.01 and selecting the top 20 most significantly up- and downregulated genes.Sequencing was done by an external facility (IKMB). After quality control with FastQC, raw reads were trimmed using Cutadapt  removing2.146 cells were harvested by scraping, resuspended in PBS, and embedded in paraffin, and 4-µm sections were stained by applying the same SOP for diagnostic immunohistochemistry assessments. The following reagents/antibodies were used for staining: hematoxylin and eosin for H&E, clone 22C3 for PD-L1, polyclonal AE1/3 for pan-cytokeratin, and clone Ber-EP4 for Ep-CAM.Per cell line, 5 × 1033.1Three patients operated on at the UMR in the years 2009–2020 and enrolled in the BioBank Rostock (BBR) presented with either primary lung tumors or brain metastases see Table 2.Routine diagnostic procedures included histological assessment of the tumor tissue, determination of the subentity, TNM classification, and grading. The primary lung tumors were further analyzed for TTF1 and PD-L1 protein expression and an NGS Illumina focus panel assessment was performed see Table 4.3.2The cells of HROBML01 and HROLu22 grow as tumor islands, and the cells possess a morphology frequently associated with epithelial tumor  cells. The cells of HROLu55 start of as large(r) cells, which reduce their size according to the space available and finally form a 100% confluent monolayer consisting of small cells in the end. Micro-photographic images of all three cell lines can be found in Cell doubling times were between 3 and 5 days. The highest doubling time with 69.31 h  was calculated for HROBML01, closely followed by HROLu55 with 72.06 h . The lowest proliferation was observed for HROLu22 with a doubling time surpassing 100 h: 101.6 h .3.3Paraffin-embedded immunohistochemical assessments performed by an expert pathologist (FP) of the three cell lines see Figure 23.4After confirmation that the cell lines represent the subentities of the tumors they were derived from, the expression of several surface markers was assessed by flow cytometry Figure 33.5In addition to the focus panel sequencing, which was performed during routine pathological assessment for the tumors of patients HROLu22 and HROLu55, we performed whole exome sequencing (WES) analyses, comparing the cell lines with the original patient tumors for all three models. For patients HROLu22 and HROLu55, tumor adjacent normal tissue was assessed in parallel. Due to the fact that HROBML01 was derived from a brain metastasis, no normal brain tissue was available.The mutational signature analysis of HROLu55 shows high similarity to cosmic signature 4, which is consistent with the mutational pattern caused by exposure to tobacco smoke Figure 4In a second step, we identified the top 30 mutated genes by the WES approach across all three tumor tissues and cell lines see Figure 5Finally, we calculated the TMB for all cancer samples (tissues and cell lines). The tumor tissue always had a higher TMB than the corresponding cell line see Table 5.3.6In addition to the WES analyses, matched RNA expression levels were assessed by RNA sequencing of the same tissue piece or cell pellet, respectively. Principal component analysis (PCA) for quality control revealed clustering of the normal tissue, the tumor tissue of HROLu22 and HROLu55 with their cell line counterparts, and a third cluster of tumor tissue and cell line for HROBML01 see 3. FurthThus, the 20 most up- and downregulated genes on RNA expression level were identified Figure 73.7A common feature of cancer cells that serves as a unit to measure the degree of aggressiveness is invasion and migration. Thus, the invasion and migration potential of the three cell lines was assessed in classical transwell assays. In comparison to the reference HROC24, the cells of cell line HROLu55 possessed a higher capacity for invasion and migration. The cells of HROLu22 and HROBML01 were comparable to the reference with regard to both invasion and migration Figure 8These properties of higher invasion and migration capacity resulted in faster wound closure rates Figure 93.8Further properties associated with tumorigenicity are the capacity to form colonies starting with very few tumor cells (≈100 cells) and three-dimensional spheroids .3.9in vitro response testing  corresponded well to the actual clinical outcome of the patients (50 of 3.66 vs. 0.73 and 0.50) and carboplatin (IC50 of 26.16 vs. 5.66 and 3.62), and at the same time, proliferation was lowest (doubling time of 101.60 h vs. 69.31 h and 72.06 h).The cell line HROLu22 represents the adenomatous type, HROBML01 represents the squamous cell type, and HROLu55 represents the pleomorphic lung cancer type. This was confirmed by an expert pathologist (FP). From the generally high level of data concordance, we conclude that these models represent the patients they are derived from very well. This is in line with what we previously already observed for models established from colorectal cancer  and gliopatients . Furtherin vitro culturing processes.One corner stone of precision medicine is the integration of NGS techniques in the molecular pathological assessments . At the Among the 30 most mutated genes, we discovered familiar cancer candidates such as TP53 and MUC16  and lungin vitro and in vivo  is the most overexpressed gene across all three of our cell lines. It is often overexpressed in lung cancer, and some studies suggest that overexpression could be linked to promoting invasive properties  and smal in vivo . Our obsin vitro, for an easier linking of NGS results to phenotypical tumor characteristics.In conclusion, we report on three PDCs established from different subentities of NSCLC including the very rare pleomorphic cell type. All PDCs retained morphological, molecular, and genetic properties of their patient tumor tissue originals. Additionally, these PDCs are a 100% pure tumor cell population and, thus, allow, besides functional analyses and response testing The raw WES and RNA-Seq Datasets presented in this article are not readily available because they contain confidential information about individuals protected under the General Data Protection Regulation. Requests to access the datasets should be directed to the corresponding author.The studies involving human participants were reviewed and approved by the ethics committee of the University Medical Center Rostock. The patients/participants provided their written informed consent to participate in this study.As principal investigator, CL designed the project and supervised experimental execution as well as data analysis. The experiments were performed and obtained data were analyzed by IA. Histological assessments were performed and analyzed by the expert pathologist FP. ML was involved in all project discussions and helped with problem solving. The manuscript was drafted by CL and IA, all authors read and approved the final version of the manuscript.This paper is dedicated to the memory of Christina S. Linnebacher, who passed away unexpectedly while this paper was being peer-reviewed. CSL was an esteemed author whose contributions to the field of oncology research were invaluable. CSL was widely known for her expertise in patient-derived cancer models, which provided crucial insights into the development and treatment of cancer. Her pioneering research will continue to have a profound impact on the scientific community, and she will be deeply missed by her family, friends, and colleagues.


Despite the significant advancements in complex anticancer therapy, the search for new and more efficient specific anticancer agents remains a top priority in the field of drug discovery and development. Here, based on the structure-activity relationships (SARs) of eleven salicylaldehyde hydrazones with anticancer activities, we designed three novel derivatives. The compounds were tested in silico for drug-likeness, synthesized, and evaluated in vitro for anticancer activity and selectivity on four leukemic cell lines , one osteosarcomic cell line (SaOS-2), two breast adenocarcinomic cell lines (MCF-7 and MDA-MB-231), and one healthy cell line (HEK-293). The designed compounds were found to have appropriate drug likeness and showed anticancer activities in all cell lines tested; particularly, two of them exhibited remarkable anticancer activity in nanomolar concentrations on the leukemic cell lines HL-60 and K-562 and the breast cancer MCF-7 cells and extraordinary selectivity for the same cancer lines ranging between 164- and 1254-fold. The study also examined the effects of different substituents on the hydrazone scaffold and found that the 4-methoxy salicylic moiety, phenyl, and pyridinyl rings are the most appropriate for anticancer activity and selectivity of this chemical class. Cancer remains the second leading cause of death worldwide, according to the World Health Organization’s released reports . More thTreatment of cancer includes a combination of chemotherapy, radiation therapy, targeted therapy, hormone therapy, immunotherapy, and bone marrow transplantation . Many diOver previous years, numerous novel physiologically active compounds have been discovered targeting specific molecular pathways involved in the development and progression of different types of cancer and providing a more targeted approach to treatment. Abemaciclib, palbociclib, and ribociclib are targeted breast cancer drugs that block the activity of two proteins on cancer cells, CDK4 and CDK6. All three drugs are approved to treat advanced or metastatic breast cancer, based on data from large clinical trials showing that they can substantially improve how long patients live without their cancer getting worse ,12,13. E2H4) and contain a C=N bond. These compounds have been found to exhibit a broad range of biological activities, including antifungal, antibacterial, and anticancer properties. Studies have shown that some hydrazones have potent anticancer effects against breast cancer and leukemia cell lines. For example, a series of hydrazones exhibit a prominent effect against MCF-7 breast cancer cells, comparable with the activity of Doxorubicin, a known medication used to treat breast cancer, bladder cancer, lymphoma, and acute lymphocytic leukemia −1 assigned to the azomethine group C=N, which proves the condensation between the aldehyde group of 4-methoxysalicylaldehyde and the amino group of hydrazides in the formation of Schiff base. The medium intensity peak around 3350–3440 cm−1 and the weak broadband at 3213–3228 cm−1 were assigned to the phenolic hydroxyl group and NH group, respectively. The intensive characteristic bands at 1640–1662 cm−1 in the spectra of the compounds were assigned to the frequency vibration of the carbonyl group C=O and suggest the existence of the hydrazones in keto form in solid state. Another important band at 1578–1600 cm−1 was attributed to n(C-NH).The structures of the newly synthesized hydrazones were found through IR and NMR spectroscopy. The IR spectra of the hydrazones show an intensive band around 1602–1609 cm1H NMR and 13C NMR spectra in deuterated dimethyl sulfoxide (DMSO). The 1H NMR spectra revealed the presence of aromatic protons in the region δ 6.50–7.81. The protons of the methoxy group appeared as a singlet at δ 3.72–3.75. Signals for the protons of the characteristic for hydrazone azomethine group HC=N- were observed at δ 8.56, 8.51, and 8.58, respectively, for compounds 12, 13, and 14. The broad singlets around δ 11.78–12.18 were assigned to the protons of the hydroxyl group from the aldehyde ring. The 13C NMR spectra demonstrated the signals corresponding to the carbon atoms of azomethine group at δ 148.87, 148.10, and 150.33, respectively, for 12, 13, and 14. The peaks at δ 162.59, 161.86, and 161.07 were assigned to the C=O group. The 1H NMR and 13C NMR data in deuterated DMSO give additional information for the new hydrazones.The hydrazones were further studied by their 50 are given in The newly synthesized derivatives were tested for cytotoxic activity on the same cell lines. Throughout the screening study, the data about the 4-methoxy hydrazones were compared with the clinically used antineoplastic drug, Melphalan (2-amino-3-[4-bis(2-chloroethyl)amino]phenylpropanoic acid). The tested compounds inhibited the proliferation of cells in a concentration-dependent manner, which enabled the construction of concentration-response curves. The experimental values for IC50 values of 0.03 μM and 0.05 μM for hydrazones 12 and 14, respectively. These were much lower (more than 900-fold) than those obtained for typical cytostatic Melphalan. The results on the acute leukemia cell line HL-60 were similar. The experimental IC50 values for compounds 12 and 14 were 0.04 μM and 0.06 μM. The cytotoxic activity of the 4-methoxy-derived hydrazones in chronic myeloid leukemia BV-173 and acute lymphoblastic leukemia KE-37 cells revealed that hydrazones produced comparable cytotoxic effects, with compound 13 being less active according to the IC50 values /IC50 (cancer cells). The SI values are given in 13 on SaOS-2) to 1254 (for compound 12 on K-562). The most selective is compound 12, with an average SI of 350, followed by compound 14, with average SI of 258. Compound 13 was the least selective, with an average SI of 14. All three new hydrazones were more selective than the positive control, Melphalan.To assess the selectivity of the newly synthesized derivatives, they were tested on the normal human embryonic kidney HEK-293 cell line. The derived ICThe findings presented here are a continuation of our previous research on salicylaldehyde hydrazones with anticancer activity ,30,31,32a values in the range of 7.67–8.08, existing at physiological pH 7.4 as 65–83% non-ionized molecules. They meet the requirements for drug and lead likeness, have small ss,VD moderate CL, and short half-lives. In terms of anticancer activity, compounds 12 and 14 are more active than compound 13 in all cell lines. The highest activity is demonstrated on the leukemic cell lines HL-60 and K-562, where the measured IC50 values are below 60 nM. Similar extreme activity is observed on the breast cancer cell line MCF-7, with IC50 of 230 nM for compounds 12 and 14. On the leukemic cell lines, both compounds are equipotent, on the triple negative breast cancer cell line MDA-MB-231, compound 14 is twice more active than compound 12 (1.22 µM vs. 2.31 µM).The designed 4-metoxysalicylic hydrazones are weak acids with pKSI. In terms of selectivity, compound 12 showed the best performance, being 1254-, 941- and 164-fold more toxic on K-562, HL-60, and MCF-7, respectively, than on normal cells. Close to it, compound 14 is the next most selective with SIs equal to 821, 685, and 179 on the same cell lines. The least selective compound was hydrazone 13.The selectivity for cancer cells of the tested compounds ranges in quite wide limits, from 2 to 1254, expressed as 12 and 14 on leukemic cells indicate that benzoyl and isonicotinoyl substituents at the Ar2 position of the hydrazone scaffold are bioisosteric. However, incorporation of a hydroxy group into the benzoyl ring (compound 13) reduced cytotoxicity between 3- (BV-173) and 292-fold (K-562). A moderate decrease is observed on sarcomic cells (15.5-fold reduction) and on breast cancer cells (1.25-fold reduction for MDA-MB-231 and 6-fold reduction for MCF-7).The close anticancer activities and selectivities of compounds Regarding the substituent at position R1, the 4-methoxysalicilic moiety was found to be the most appropriate for anticancer activity compared to 3-methoxy, 5-bromo, and 5-nitro-substituted salicylic hydrazones.Overall, this study provides valuable insights into the design and development of new hydrazone-based compounds with potential anticancer activity. The results of this study clearly guide further research into hydrazone derivatives as potential and selective anticancer agents and contribute to the development of more effective cancer treatments.50 values on the following cell lines: HL-60, KE-37, K-562, BV-173, SaOS-2, MDA-MB-231, and MCF-7. For the purposes of SAR analysis, the IC50 values were converted in p-units (−logIC50) and ligand efficiencies (LEs) were calculated according to the formula: LE = pIC50/n, where n is the count of non-hydrogen atoms in the molecule.The training set of salicylaldehyde hydrazones contained 11 compounds synthesized and tested before in our Lab ,30,31,32uf; unbound clearance, uCL; and steady state volume of distribution, ssVD. The datasets consisted of 132 acidic drugs, extracted from Obach’s database–the largest and best curated source of data for the key pharmacokinetic parameters after iv administration +. Calculated for C15H14N2O3: C, 66.66; H, 5.22; N, 10.36. Found: C, 67.02; H, 5.07; N, 10.44.Yield: 84%; mp: 182–183 °C; Color: Pale yellow; IR : 3400 (OH), 3300 (OH), 3213 (NH), 1662 (C=O), 1608 (C=N), 1589 (C-NH). 1H NMR  δ ppm: 3.77 , 6.50 , 6.51 , 7.38 , 6.87 , 7.81 , 8.51 , 10.13 , 10.13 , 11.78 ; 13C NMR  δ ppm: 55.26 (OCH3), 112.52, 115.07, 117.18, 117.86, 118.95, 123.27, 129.72, 148.10 (CH=N), 151.39, 152.08, 160.84, 161.86 (C=O). HR ESI–MS m/z: 287.10223 [M + H]+. Calculated for C15H14N2O4: C, 62.93; H, 4.93; N, 9.79. Found: C, 62.98; H, 5.07; N, 9.68.Yield: 80%; mp: 216–217 °C; Color: Pale yellow; IR : 3350 (OH), 3190 (NH), 1658 (C=O), 1609 (C=N), 1600 (C-NH). 1H NMR  δ ppm: 3.78 , 6.51 , 6.53 , 7.47 , 7.83 , 8.58 , 8.78 , 11.43 , 12.18 . 13C NMR  δ ppm: 55.30 (OCH3), 111.77, 117.31, 118.64, 118.93, 121.49, 140.04, 150.33 (CH=N), 150.33, 151.50, 152.16, 161.07 (C=O). HR ESI–MS m/z: 272.10267 [M + H]+. Calculated for C14H13N3O3.H2O: C, 58.13; H, 5.23; N, 14.53. Found: C, 58.47; H, 5.27; N, 14.49.Yield: 97%; mp: 232–233 °C; Color: Bright yellow; IR , SKW-3 (KE-37 derivative) (T-cell leukemia), BV-173 and K-562 (chronic myeloid leukemia), MDA-MB-231 (ER-negative breast carcinoma), MCF-7 (ER-positive breast adenocarcinoma), and SaOS-2 (osteogenic sarcoma). The cells were provided from the German Collection of Microorganisms and Cell Cultures . The solid tumor cell lines  were incubated in 90% RPMI-1640 supplemented with 10% fetal bovine serum (FBS), non-essential amino acids, 1 mM sodium pyruvate, and 10 mg/mL human insulin as monolayer adherent cultures. The remaining cell lines were cultivated as a suspension culture in cell culture flasks at 37 °C in an incubator “BB 16-Function Line” Heraeus  with humidified atmosphere and 5% CO5 cells per ml in 96-well flat-bottomed microplates (100 μL/well), and after 24 h of incubation at 37 °C, they were treated to varied doses of the tested substances for 72 h. At least eight wells were used for each concentration. Following the incubation with the test compounds, aliquots of 10 μL MTT solution (10 mg/mL in PBS) were applied to each well. The microplates were then incubated at 37 °C for 4 h before the MTT-formazan crystals were dissolved with 100 μL/well 5% HCOOH in 2-propanol. The MTT-formazan absorbance was measured at 580 nm using a microprocessor-controlled microplate reader (Labexim LMR-1).The cytotoxic activity of the examined substances was determined using Mossman’s  MTT [3- of at least eight independent experiments. The IC50 values were obtained by non-linear regression analysis from the concentration-response curves and represent the concentrations of the tested compounds causing a 50% decrease in cell survival.The cell survival data were standardized as a percentage of the untreated control (set as 100% viability). The biological data were processed statistically using Student’s t-test, where values were considered statistically significant at