We analyzed the epigenomic consequences of rearrangements between the super-enhancers of the immunoglobulin heavy locus (IGH) and proto-oncogene CCND1 that are common in B cell malignancies. By integrating BLUEPRINT epigenomic data with DNA breakpoint detection, we characterized the normal chromatin landscape of the human IGH locus and its dynamics after pathological genomic rearrangement. We detected an H3K4me3 broad domain (BD) within the IGH locus of healthy B cells that was absent in samples with IGH-CCND1 translocations. The appearance of H3K4me3-BD over CCND1 in the latter was associated with overexpression and extensive chromatin accessibility of its gene body. We observed similar cancer-specific H3K4me3-BDs associated with hijacking of super-enhancers of other common oncogenes in B cell  and T cell malignancies . Our analysis suggests that H3K4me3-BDs can be created by super-enhancers and supports the new concept of epigenomic translocation, in which the relocation of H3K4me3-BDs from cell identity genes to oncogenes accompanies the translocation of super-enhancers.Chromosomal translocations are important drivers of haematological malignancies whereby proto-oncogenes are activated by juxtaposition with enhancers, often called  The human CH region encodes nine different Ig isotypes: IGHA2, IGHE, IGHG4, IGHG2, IGHA1, IGHG1, IGHG3, IGHD, and IGHM . Complex regulatory and genomic rearrangements in the IGH locus are required to ensure that Ig transcripts containing only one of each of these gene segments are expressed in each B cell at the correct stage of B cell differentiation. These natural processes predispose human Ig loci to inappropriate translocation events. As a result, these super-enhancer-rich regions are commonly hijacked in B cell malignancies, providing the optimal model in which to address whether H3K4me3-BDs are formed as a consequence of super-enhancer activity.The immunoglobulin (Ig) loci contain powerful super-enhancers to drive antibody formation and expression. The immunoglobulin heavy . We defined H3K4me3-BDs as domains with H3K4me3 >2 kb and compared them across different hematopoietic cell types . We observed shared H3K4me3-BDs in all of the cell types that were associated with basic cellular functions . Only a small proportion of detected H3K4me3-BDs were cell type–exclusive , identifying cell-identity genes  as reported by the other studies mentioned above. With recent studies observing increased interactions between H3K4me3-BD and super-enhancers . The cell type–exclusive H3K4me3-BDs  had proximal super-enhancer(s) more frequently than cell type–nonexclusive H3K4me3-BDs .H3K4me3-BDs have been defined in different ways, using size- and height-ranked . Howevernhancers , we alsoCCND1) at 11q13 and IGH at 14q32 (IGH-CCND1). To begin to understand how this translocation event leads to CCND1 activation, we characterized the epigenomic landscape in these two regions from healthy and malignant B cells. To precisely define the location and activity of the human IGH super-enhancers and promoters and the chromatin dynamics of the CCND1 locus in B cells, we used 108 chromatin state maps for different hematopoietic cell types built with over 700 ChIP-seq data sets from BLUEPRINT , Chr 14: 106,144,200–106,179,400 (Eα1), and Chr 14: 106,281,800–106,326,200 (Eμ). The Eμ enhancer showed dynamic changes associated with the B cell development stage. A larger region appears active in naive B cells before entering the germinal center (GC), whereas in post-GC B cells, the region of activity is reduced (class-switched memory B cells and plasma cells)  were found within Ca cells) . A simila cells) . None ofCCND1 is one of the most common IGH translocation partner genes in hematological malignancies, a hallmark of MCL  samples show a similar pattern to healthy B cells, with the Polycomb state domain upstream of the active promoter. DLBCL and BL samples show a narrow H3K4me3 active promoter but lack the upstream Polycomb domain. All MCL and some MM samples have a larger active promoter/enhancer region extending upstream of and downstream from the transcription start site, with most of the gene body containing H3K4me3 marks, a pattern that looks comparable to an H3K4me3-BD .We discovered a B cell–specific H3K4me3-BD at the genomic location, Chr 14: 106,346,800–106,387,800 (41 kb), overlapping the DGH locus . This elIGH translocations, we studied the MM cell line U266, in which the IGH super-enhancer Eα1 is inserted next to the CCND1 proto-oncogene . We confirmed two chromosomal breakpoints that occur in the IGHE and IGHA switch regions and result in the cut and paste of the IGH Eα1 super-enhancer into Chromosome 11, ∼12 kb upstream of CCND1 . This suggests that the inserted Eα1 super-enhancer has a bidirectional effect, generating an H3K4me3-BD over another adjacent gene.To further investigate the presence or absence of H3K4me3-BD as a result of oncogene . This ceof CCND1 . The vas66 cells . Importaene body . This ob samples , 2. This samples , as obse samples , as obseSupplemental Table S3). Two of the cell lines had an IGH-CCND1 rearrangement, including U266  and the MCL cell line Z-138. We observed the H3K4me3-BD and strong DNase I hypersensitivity signals encompassing the entire coding region of the CCND1 gene in both cell lines  and seven MM patients whose diagnostic samples were engrafted in murine models (patient-derived xenografts [PDXs]). Each cell line had a complex rearrangement identified by targeted sequencing of the IGH locus involving multiple proto-oncogenes . 10x Genome sequencing detected in patients P1 and P2 the second most common IGH translocation in MM, t, involving the transcription factor MAF. Patients P3 and P4 had IGH translocations t, involving FGFR3 and NSD2, with the remaining three patients (P5–P7) having no detectable translocation involving any of the Ig loci . No cell lines or patients had an IGH-CCND1 rearrangement detected by targeted or genome sequencing; therefore, as expected, we did not observe an aberrant H3K4me3-BD or an increase in mRNA transcript levels for CCND1 in these samples . We observed a Polycomb chromatin state over MYC, but the analysis of each histone modification separately identified an H3K4me3-BD over MYC in the two cell lines , possibly reflecting repression of one allele of MYC and activation of the other owing to being juxtaposed to an IGH super-enhancer (In the two cell lines and four of the patient samples (P1–P4), we observed an H3K4me3-BD over the coding region of  RNA-seq B. This wenhancer .MAF; however, we see overexpression of MAF in association with an H3K4me3-BD .In the remaining two patients , we were unable to detect a genomic rearrangement of a super-enhancer in close proximity to K4me3-BD B. These IGH super-enhancer. Although the chromatin state model  are also regulated by super-enhancers, undergo similar inherent somatic rearrangement events, and are involved in translocation events in T cell acute lymphoblastic leukemia (T-ALL) . The Polycomb chromatin state was observed in healthy early and late cortical T cells and CD4 and CD8A expressing T cells . To provide mechanistic data to support the generation of H3K4me3-BD by super-enhancer relocation, we used our previously CRISPR-Cas9 engineered T-ALL cell line PEER, which contains the same 12-bp insertion upstream of the TAL1 gene, resulting in TAL1 overexpression .Next, we wanted to assess whether de novo super-enhancers, generated by somatic nucleotide insertions, could also generate aberrant H3K4me3-BD. An aberrant H3K4me3-BD was observed across the e Jurkat C, where e Jurkat . No supepression . H3K4me3In summary, we found that the appearance of H3K4me3-BDs is associated with cancer-specific super-enhancer activation in both B cell– and T cell–derived hematological malignancies. Our data suggest that H3K4me3-BDs could be a signature of super-enhancer targets in general and a useful marker to identify deregulated genes affected by the hijacking of super-enhancers in cancer.CCND1 at 11q13 and the IGH locus at 14q32. We have shown that genomic relocation of the IGH Eɑ1 super-enhancer alters the location of an H3K4me3-BD in rearranged malignant B cells. The presence of broad H3K27ac domains over CCND1 have previously been associated with t translocations in MM cell lines including U266 . We also observed H3K4me3-BDs over additional proto-oncogenes rearranged in B cell and T cell malignancies, including the involvement of gene-specific and de novo super-enhancers. H3K4me3-BDs are associated with high levels of stable transcription of cell-identity and tumor suppressor genes  and chromatin remodelers (NSD2), it may not be surprising that the increased expression and presence of the translated protein could indirectly drive the appearance of H3K4me3-BD over other cancer-associated genes.We analyzed a total of 14 hemato-oncology samples, newly generated data for seven PDX samples, and seven patient-derived cell lines. Twelve of these cases were positive for a genomic abnormality involving hijacking of super-enhancers and proto-oncogene activation (the remaining two did not have these rearrangements). All 12 samples showed an H3K4me3-BD over the oncogene specific to a genomic rearrangement, confirming our hypothesis about the association of H3K4me3-BD and super-enhancers. In three of these 12 samples, additional H3K4me3-BDs were observed over other oncogenes not involved in a genomic rearrangement; however, there was no evidence of super-enhancer translocations at these loci. This could be because of unidentified chromosomal abnormalities involving other super-enhancers or by the existence of additional mechanisms associated with H3K4me3-BD formation. This is in agreement with our complementary publication by epigenomic translocation, a consequence of hijacking of super-enhancers. In this model, a super-enhancer-driven wild-type H3K4me3-BD “relocates” to a target proto-oncogene as the result of a genomic rearrangement, leading to oncogene activation , where the hijacked oncogene and super-enhancer reside . This data set was composed of 85 samples from healthy donors  and 23 samples from patients (16 primary and seven cell lines) with different B cell hematological malignancies. Five of these cell lines had DNase I hypersensitivity and RNA-seq data available . BLUEPRINT data were downloaded from ftp://ftp.ebi.ac.uk/pub/databases/blueprint/ paper_data_sets/chromatin_states_carrillo_build37 (ChIP-seq chromatin states) and ftp://ftp.ebi.ac.uk/pub/databases/blueprint/data/homo_sapiens/GRCh37 (DNase I hypersensitivity and RNA-seq). Early and late cortical T cell ChIP-seq chromatin states are accessible from GitHub (https://github.com/guillaumecharbonnier/mw-cieslak2019/tree/master/src/hub). Detailed laboratory methods and data processing of BLUEPRINT experiments are described elsewhere  were used  and CCND1 (promoter and Polycomb) loci was built using 15 BLUEPRINT B cell/plasma-cell samples derived from healthy donors . Epigenomic consensus of super-enhancers and H3K4me3-BDs at TRA/TRD and BCL11B loci was assembled using 17 BLUEPRINT T cell samples derived from healthy donors. MCL (JVM-2 and Z-138) and MM (U266) cell lines with known IGH-CCND1 rearrangements  were used to determine the relocated CCND1 H3K4me3-BD.Epigenomic annotation of IGH (overlapping the CH and IGH intronic region), TRA/TRD (constant and joining regions), and BCL11B (from the gene position up to ∼1 Mb downstream) loci. Promoters were determined as a segment >2.5 kb within the expected promoter positions of IGH (CH and intronic region) and CCND1 genes. H3K4me3-BDs were scanned at IGH CH, JH and DH, and CCND1, TRA/TRD, and BCL11B loci, with a minimum size of 15 kb. Polycomb at the CCND1 locus was obtained as regions upstream and overlapping with the promoter.Segmentation files of relevant samples were filtered for the subset of chromatin states  of interSupplemental Tables S2, S3). For this analysis, the proximity of H3K4me3-BDs (≥2 kb) to super-enhancers was compared (≥5 kb) to a control group of small promoters . Super-enhancers overlapping directly H3K4me3-BD and/or genes with H3K4me3-BD were excluded from the analysis. Also, promoters in genes with H3K4me3-BD in alternative transcription start sites were excluded. Genomic distances between H3K4me3-BD/promoters and super-enhancers up to 5 Mb were analyzed by 100-kb bins. Gene enrichment for biological processes was performed using R package clusterProfiler v3.18.0  were sequenced using targeted high-throughput sequencing covering the following genomic regions: (1) 4.2 Mb, extended coverage for chromosomal abnormality detection within IGH, IGK, IGL, and MYC (4.2 Mb); and (2) 0.6 Mb, exonic regions of 127 myeloma-specific genes for mutation analysis and 27 additional regions for efficient data normalization. GRCh37 human genome assembly was used for sequence mapping using BWA-MEM (v0.7.12). Chromosomal rearrangements were called using Manta v.0.29.6 .PDXs were generated by passaging primary patient CD138 + selected cells through the previously described SCID-rab myeloma mouse model . DetaileSupplemental Table S5) were manually inspected.DNA from seven PDX samples were sequenced using phased whole-genome sequencing (10x Genomics) at Hudson Alpha. Long ranger (10x Genomics) pipelines were used for data processing, including alignment to GRCh38 genome assembly and structural variant calling. Germline controls were used to distinguish somatic abnormalities, and chromosomal breakpoints of detected translocations , the model used in the BLUEPRINT data set . Additio2 expression calculated.RNA-seq was performed using 100 ng total RNA with genomic DNA removal using the TURBO DNA-free kit (Ambion). RNA was prepared using the TruSeq stranded total RNA Ribo-Zero gold kit (Illumina), and libraries were sequenced using 75-bp paired-end reads on a NextSeq 500 (Illumina). RNA-seq data were analyzed as previously reported . Brieflyhttps://www.ncbi.nlm.nih.gov/geo/) accession numbers GSE54379 and GSE65687, were used for histone modification analysis of three T-ALL cell lines KOPT-K1, DND-41, and Jurkat . Raw files were downloaded using the associated NCBI Sequence Read Archive  accession and converted to FASTQ files using NCBI fasterq-dump (SRA Toolkit v2.9.6-1). Appropriate sequencing runs were merged, and sequences were mapped to the GRCh37 genome assembly using BWA (v0.7.17), followed by processing to MACS2 (v2.2.5) (http://dcc.blueprint-epigenome.eu/). Histone marks H3K4me1, H3K4me3, H3K27me3, H3K27ac, and H3K36me3 in all three cell lines were used in this study, with the exception of H3K27me3 in Jurkat as this was not available.Publicly available ChIP-seq data, accessed under the NCBI Gene Expression Omnibus   peaks, uhttp://dcc.blueprint-epigenome.eu/#/md/methods). Prepared libraries were sequenced in paired-end 50 + 30 bp mode using the NextSeq 500/550 (Illumina) according to the manufacturer's instructions. FASTQ files were processed by BLUEPRINT pipeline as T-ALL cell lines above. Raw read counts were collected using the Genomic Analysis Toolkit v4.2  PEER cell lines were included. The genome editing method was published elsewhere . ChIP-sekit v4.2  in 200-bP-values < 0.05 were considered statistically significant. Figures involving data and annotation alignment to the human genome were generated using karyoploteR (v1.16.0) R package  under accession number PRJNA635269. All raw and processed ChIP-seq data for the KMS11 and MM1S myeloma cell lines, as well as the PEER T-ALL cell line, have been submitted to the NCBI Gene Expression Omnibus  under accession number GSE151556. All high-throughput sequencing data for the MM PDXs have been submitted to the European Genome-phenome Archive (https://ega-archive.org/) under accession number EGAS00001005684.The DNA sequencing data for the U266, KMS11, and MM1S myeloma cell lines generated in this study have been submitted to the NCBI BioProject database (


Objective: Erchen Decoction (ECD), a well-known traditional Chinese medicine, exerts metabolism-regulatory, immunoregulation, and anti-tumor effects. However, the action and pharmacological mechanism of ECD remain largely unclear. In the present study, we explored the effects and mechanisms of ECD in the treatment of CRC using network pharmacology, molecular docking, and systematic experimental validation.Methods: The active components of ECD were obtained from the TCMSP database and the potential targets of them were annotated by the STRING database. The CRC-related targets were identified from different databases . The interactive targets of ECD and CRC were screened and the protein-protein interaction (PPI) networks were constructed. Then, the hub interactive targets were calculated and visualized from the PPI network using the Cytoscape software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. In addition, the molecular docking was performed. Finally, systematic in vitro, in vivo and molecular biology experiments were performed to further explore the anti-tumor effects and underlying mechanisms of ECD in CRC.Results: A total of 116 active components and 246 targets of ECD were predicted based on the component-target network analysis. 2406 CRC-related targets were obtained from different databases and 140 intersective targets were identified between ECD and CRC. 12 hub molecules  were finally screened from PPI network. GO and KEGG pathway enrichment analyses demonstrated that the biological discrepancy was mainly focused on the tumorigenesis-, immune-, and mechanism-related pathways. Based on the experimental validation, ECD could suppress the proliferation of CRC cells by inhibiting cell cycle and promoting cell apoptosis. In addition, ECD could inhibit tumor growth in mice. Finally, the results of molecular biology experiments suggested ECD could regulate the transcriptional levels of several hub molecules during the development of CRC, including MAPKs, PPARs, TP53, and STATs.Conclusion: This study revealed the potential pharmacodynamic material basis and underlying molecular mechanisms of ECD in the treatment of CRC, providing a novel insight for us to find more effective anti-CRC drugs. Colorectal Cancer (CRC) is one of the most commonly diagnosed malignant tumors and is a leading cause of tumor-related mortality worldwide . In 2020In recent years, the value of Traditional Chinese medicine (TCM) in tumor treatment has been high-profile based on its low toxicity and multi-target activity . Severalin vitro and in vivo experiments were utilized to verify the above molecules screened by network pharmacology. This study will explore the function of ECD for CRC treatment, understand their underlying anti-tumor mechanisms, provide evidence for the application of ECD in tumor therapy, and open new horizons for therapeutic intervention in the future.TCM is characterized by multiple targets and network pharmacology is a systematic method that combines molecular biology, bioinformatics, and computer science into the pharmacological study of TCM to explore their underlying mechanisms . In thisGlycyrrhiza glabra”, “Citrus reticulata”, “Pinellia ternata” and “Poria cocos” were retrieved from the TCM Systems Pharmacology Database  (https://cn.string-db.org) (The keywords “msp.php) . Oral bi-db.org)  was used“Colorectal Cancer” was used as the keyword to search the CRC-related targets in the online Mendelian inheritance in man (OMIM) database , the datThe intersective targets were obtained between the ECD components and CRC disease targets. Then, these targets were imported into the STRING database, and the highest confidence was set as 0.9 to show the PPI network. PPI network was visualized in Cytoscape 3.9.1  softwareThe active components in ECD and the interaction targets were constructed to the active components-targets-CRC network using Cytoscape 3.9.1 software. In addition, the top five components in the Degree value of ECD were selected to proceed molecular docking.https://pubchem.ncbi.nlm.nih.gov/) (https://www.rcsb.org/) (via the DeepSite database (PubChem database (ih.gov/)  was applsb.org/) . The mecdatabase  and Autodatabase  was utildatabase .https://david.ncifcrf.gov/) (https://www.omicshare.com/tools) tools. The GO enrichment analysis includes biological processes (BP), cellular component (CC), and molecular function (MF).The GO  and KEGGrf.gov/)  and Omicrf.gov/)  (https:/Pinellia ternata (34.5%), Citrus reticulata (34.5%), Poria cocos (20.7%), and Glycyrrhiza glabra (10.3%). The ECD granules were dissolved to 0.2 g/ml in phosphate buffered saline (PBS) and filtered through a 0.22 μm pore size filter . Then the ECD filtrate was divided into several centrifuge tubes and stored at −20°C for future use.Granules of ECD were purchased from Guoyi hospital, Fujian University of TCM , composed of 2.The human colorectal cancer cell lines HT29, SW620, and DLD-1, were obtained from the American Type Culture Collection (ATCC). Consistent with the previous methods , all cel50) values of each cell line based on different concentrations and times of ECD treatment were determined using nonlinear regression. Finally, the cell viability of each cell line with low (IC25), medium (IC50), and high concentrations (IC75) of ECD treatment was also further tested in different periods.The working concentrations of ECD were diluted in complete medium to 0, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, 40, and 80 mg/ml. Three wells (repeats) were applied for each concentration and repeated at least 3 times. After the gradient concentrations of ECD treatments for 24 h, 48 h, or 72 h, cell viability assays were measured by Cell Counting Kit 8 . Then, the half-maximal inhibitory concentration .4 cells per well and cultured for 24 h. Cells were treated with the low (IC25), medium (IC50), and high concentrations (IC75) of ECD for 48 h. Then, 10 μM EdU  was added and incubated for 2 h. The remaining operations, including fixing, glycine incubating, and dilution, were performed based on the standard protocols in a light-repelled environment. The fluorescence images of these cells were captured under an inverted fluorescence microscope  and at least three random fields in each well were photographed.Three CRC cell lines were all plated on 6-well plates at 2 × 1025), medium (IC50), and high concentrations (IC75) of ECD for 48 h. For the cell cycle assay, the cells (at least 1 × 106) were fixed by 75% ethanol and stored at 4°C overnight. After washing twice with cold PBS, 300 μl PI/RNase staining  was added to each sample and incubated for 30 min at 37°C under protection from light. The cell cycle was then analyzed by flow cytometry . Similarly, for the cell apoptosis assay, both floating and attached cells in each well were harvested and washed twice with cold PBS. Then, cells were incubated with 3 μl FITC-Annexin V  and 5 μl PI  in 100 μl binding buffer at 37°C under protection from light. Next, 200 μl binding buffer was added to each sample, and cell apoptosis was also measured by flow cytometry.Three CRC cell lines were all plated and cultured on 6-well plates for 24 h and treated with the low . Seven days after cell injection, mice were randomly divided into four groups: control , ECD-L, ECD-M, and ECD-H . ECD was dissolved in a PBS solution and delivered daily by intragastric administration for 14 days. Then, tumor volumes were measured every 3 days and calculated by the formula: length × width2/2. At the end of the experiment, mice were sacrificed and tumors were photographed, weighed, and stored. H and E staining and Immunohistochemistry (IHC) of the paraffin-embedded tumor tissue sections were performed based on the standard protocols. Antibodies for IHC: p21  and p38 MAPK .Nude mice  were purchased from Shanghai Jihui Laboratory Animal Care Co., Ltd. (China). All animal experiments in this study were conducted based on the Community guidelines of the animal care and use and were approved by the Ethics Committee of Fujian University of Traditional Chinese Medicine (No. 3501020096434). For the construction of the subcutaneous xenograft mouse models, 3 × 10p < 0.05 was regarded as a statistically significant difference.All the statistical analysis was performed by the R (version: 3.4.0) and SPSS (version: 25) software for double checking in this study. All experiments were repeated more than three times, and the corresponding results were presented as means ± SD. Student t and one-way ANOVA tests were used to compare two or more groups. Categorical variables were analyzed by chi-square test. A schematic flow chart of the study was shown in 2406 CRC-related targets were obtained from four different databases, including OMIM, DisGeNet, GeneCards, and DrugBank. Besides, these targets from different databases were screened and deduplicated. A Venn diagram  was drawvia four algorithms and shown in To further explore the relationship among the 140 intersective target molecules, the PPI network was constructed by the STRING database, containing 132 nodes and 690 edges, as shown in To verify the correlation between ECD and CRC, molecular docking, a novel method, was performed by PyMOL software. The top five main active components were screened by Degree value, including Quercetin (Que), Naringenin (Nar), Licoarylcoumarin (Lic), Kaempferol (Kae), and Vestitol (Ves), displayed as 2D structure in To further explore the above molecules of the PPI network, a bubble diagram was drawn to show the results of GO and KEGG enrichment analysis concisely. The top 20 enrichment processes of GO enrichment analysis were shown in 50 values of each cell line suggest that ECD could significantly inhibit the proliferation of CRC cells in a time- and dose-dependent manner. Considering the time-preference effects of ECD, the compromised duration (48 h) has been chosen to conduct the follow-up experiments. In this study, the IC50 values for HT29, SW620, and DLD-1 cell lines were 14.80 ± 1.00 mg/ml, 14.37 ± 0.48 mg/ml and 15.19 ± 3.13 for 48 h. CCK-8 assay, colony formation assay, and EdU assays were utilized to investigate the effects of ECD on the proliferation of CRC cell lines. CCK-8 assays further showed the cytotoxicity of ECD in manners of dose- and time-dependence  were selected and treated with different working concentrations of ECD (0–80 mg/ml) for 24, 48, and 72 h respectively. As shown in pendence . ECD coupendence  and redupendence  in a dospendence . The mRNll lines . These rWith the increasing concentrations of ECD, the significant reductions in the number of CRC cell lines, abnormal cell morphology , and increasing floating cells and cell debris were observed from the light microscopy images . These fAccording to the results of network pharmacology, several key molecules  and pathways  were predicted to be underlying molecular mechanisms of ECD in CRC treatments. Surprisingly, the results of RT-PCR showed that ECD could regulate the transcriptional levels of MAPKs , PPARs , TP53, and STATs (STAT1 and STAT3) in each CRC cell line . After tin vivo, the HT29 cell xenograft tumor models were established. The results showed that ECD could significantly inhibit tumor growth in a dose-dependent manner  in TME . StudiesConsidering over-reliance on the public datasets and bioinformatics technology could destabilize the results, a systematic experimental validation was performed. In our study, ECD showed certain antiproliferative effects on CRC cells in manners of dose- and time-dependence by cell viability assay, colony formation assay, and EdU incorporation assay. In addition, our results indicated that ECD induced cell cycle arrest in the S phase of CRC cells, down-regulated the transcriptional levels of CDK1 and CDK6, and up-regulated the transcriptional levels of CDKN1A. Cyclin-dependent kinases (CDKs) are reported as cell cycle regulators and the abnormal activation of them can accelerate tumor cell proliferation . CDKN1A via the STAT3/Cyclin D1 pathway and it was the first study on the mechanism of ECD in the treatment of tumors (In spite of the fact that ECD has been widely used to treat many diseases in China, very few studies have been conducted to investigate its mechanisms of action. A previous study has shown that ECD provides therapeutic effects on obesity by regulating the gut microbiota in rats . The stuf tumors . In the f tumors . ConsistIn conclusion, based on previous studies, we have explored and supplemented several potential mechanisms of ECD in the treatment of CRC, providing biological evidence for the clinical application of ECD in tumor therapy. In addition, we also found that ECD may be related to the immunotherapy and targeted therapy for CRC based on our results . However


Additionally, we introduce a novel approach to derive an approximate value of the MCF MKM model parameters by knowing only the animal species and the mean number of chromosomes. The overall good agreement between MCF MKM predictions and in vitro data suggests the MCF MKM can be reliably used for the RBE calculations. In most cases, a reasonable agreement was found between the MCF MKM and the LEM IV.The relative biological effectiveness (RBE) calculations used during the planning of ion therapy treatments are generally based on the microdosimetric kinetic model (MKM) and the local effect model (LEM). The Mayo Clinic Florida MKM (MCF MKM) was recently developed to overcome the limitations of previous MKMs in reproducing the biological data and to eliminate the need for ion-exposed in vitro data as input for the model calculations. Since we are considering to implement the MCF MKM in clinic, this article presents (a) an extensive benchmark of the MCF MKM predictions against corresponding in vitro clonogenic survival data for 4 rodent and 10 cell lines exposed to ions from  The moddel LQM ,18) are  are ß) odel LQM ,19. . ß of thWhile amorphus track structure models such as the LEM  rely on Despite some limitations of the LEM I  were repß) of the LQM [The original version of the MKM , develop the LQM ,11,31. ß by using whole spectral information. Additionally, the mean radius of the radiation-sensitive subnuclear domains is a priori calculated using a new methodology based on the population-mean amount of DNA content in the cell nucleus [Rn, mean radius subnuclear domains rd), the MCF MKM was successful in predicting average and experiment-specific RBE trends for the most commonly used mammalian cell line  and the clinically relevant human salivary gland tumor cells (HSG cell line) for ions from 1H to 132Xe. A sensitivity test proved that the a priori determined MCF MKM parameters for these two cell lines were in good agreement with the ones obtained by fitting the in vitro data [In view of the upcoming integrated proton-carbon ion therapy center at Mayo Clinic Florida , a nove nucleus . This istro data . 1H to 238U. Since morphologic measurements of the cell nucleus (Rn) are not always available in the literature, this work also presents a phenomenological approach to derive an approximate value of both model parameters  by knowing the animal species and, for aneuploid cancer cells, the mean number of chromosomes. When possible, the MCF MKM prediction were compared with published results of the LEM IV [Considering a possible clinical implementation of the MCF MKM, it is of primordial importance to (a) validate the model predictions against a large dataset of in vitro clonogenic survival data for various cell lines and (b) compare the MCF MKM results with the results of the most recent version of clinically relevant models such as the LEM. Therefore, in this article we investigate the accuracy of the MCF MKM in systematically predicting the LQM terms and the RBE values for 4 rodent  and 10 human repair-competent cell lines in case of exposures to ions from e LEM IV . ß) and the RBE for a surviving fraction of 10% (RBE10%) predicted by the MCF MKM are compared with the experimental in vitro data from PIDE 3.2 [1H and 2H ions) were pooled together since negligible differences were observed when the results are plotted as a function of the LET [In the following paragraphs, the linear and quadratic terms of the LQM (α and PIDE 3.2  and publPIDE 3.2  with thePIDE 3.2 ). Since  the LET ,36.10% for better clarity in the comparison between the three data series . Nonetheless, examples of RBE calculations for other surviving fractions (50% and 1%) are given in Though the MCF MKM can be used to compute the RBE also for other surviving fractions, the plots are limited to the RBEß [The PIDE in vitro database does not provide uncertainty intervals for the clonogenic survival data. This is due to the fact that most of the published biological studies do not include an uncertainty analysis and, in some cases, not even an indication of the statistical dispersion of the results. The RBE results for the clonogenic survival essay are affected by several sources of uncertainty, such as: the different operational protocols between different groups , cell lß ). All ofß , is to bß ,36) and ß . Consequ1H ions where the LEM IV seems to predict higher α and RBE10% values, the results of the calculations with the two models are in good agreement between each other and with the in vitro data of 1H to 20Ne). It is worth remembering that the prediction of large ß values  is beyond the capabilities of both models. Furthermore, it should be noted that, here and in the following, the ß values predicted by the LEM show a sharper decrease with the increase of the LET with respect to the ß values calculated with the MCF MKM. In case of 28Si and heavier ions  are shown in ier ions , the agr40Ar ions. In this case, the MCF MKM predictions appear to be higher than the in vitro data. No LEM IV calculations for the CHO cell line were available for 40Ar ions. The somehow counterintuitive increase of the LEM-calculated ß term at very high LET is due to the current implementation of the DNA damage enhancement factor [The MCF MKM and the LEM IV results appear to be in good agreement with the in vitro data for chinese hamster ovary cells (CHO and CHO-K1 cell lines) over the whole investigate ion-LET range . The onlt factor .1H and 7Li ions. Except for the larger in vitro ß data point for 26 keV/µm protons , the model calculations  are in good agreement with the in vitro data for both 1H and 7Li ions. Due to the approach used in Equation (4), the MCF MKM always predicts a monotonic decrease of ß with the increase of the particle LET . The MCF MKM predictions for transformed mouse epidermal cells (PDV cell line) are plotted in ß value around 160 keV/µm, this has a little effect on the calculated RBE10% since the α term is significantly larger than ß. As for α, the RBE10% calculated with both models are similar and in good agreement with the experimental data.The α values predicted by MCF MKM and the LEM IV for carbon-irradiated rat prostatic adenocarcinoma epithelial cells  overestimate the in vitro α values for 1H ions. The overestimation is significantly larger for the LEM IV. By contrast, the proton ß values calculated by the two models agree well between each other but underestimate the in vitro ß data. This concomitant overestimation of α and underestimation of ß might be an artifact due to the anti-correlation between the two LQM terms during the fit of the in vitro data [10% values predicted by the MCF MKM are in good agreement with the corresponding in vitro results for 1H ions. By contrast, the LEM IV seems to overestimate the HeLa proton RBE10%. Though the results of the three data series  are in closer agreement, the discussion of the HeLa results for 4He ions follows the one for 1H ions and it is not repeated. The results of human cervical cancer cells (HeLa cell line) are plotted in tro data ,45. None10% calculated by the two models (MCF MKM and LEM IV) in case of proton-irradiated human fetal lung fibroblasts (HF19 cell line) are higher than the corresponding in vitro data . 12C ions. No LEM IV data were available. A good agreement between the calculated and the measured cell response is present over the whole LET range. The MCF MKM results for human mammary epithelial cells (M/10 cell line) are shown in 12C, 20Ne, and 28Si ions. For 12C ions, the α and RBE10% values calculated with the MCF MKM and the LEM IV are reasonably similar and in good agreement with the in vitro data. The MCF MKM accurately describes the α and the RBE10% trends for 20Ne ions. The α and RBE10% values calculated by the MCF MKM for 28Si ions appear to underestimate the in vitro data. The LEM IV appears to systematically underestimate α and RBE10% for both 20Ne and 28Si ions. Once the dispersion in the in vitro ß values for 12C and 20Ne ions is considered, we conclude that both models describe this quantity reasonably well. On the other hand, though the ß values calculated by the MCF MKM and LEM IV for 28Si ions are somehow similar, both models significantly overestimate the experimental trend of ß.1H ions, the MCF MKM and the LEM IV seem to overestimate the in vitro α and RBE10% values. The overestimation is larger for the LEM IV. Except for the two highest-LET in vitro entries for 12C ions, the MCF MKM seems to well reproduce the in vitro α values. On the other hand, the LEM IV results appear to be smaller. However, the LEM IV seems to better describe the ß trend as a function of the LET for 12C ions. The in vitro RBE10% of 12C with LET < 100 keV/µm appears to be underestimated by both models, with the LEM IV showing the smaller RBE10% values. The MCF MKM seems to reproduce the in vitro α entry for 40Ar ions reasonably well. Though larger than the corresponding in vitro entry, both models predict a similar ß value for 40Ar ions. The MCF MKM and the LEM IV, respectively, overestimates and underestimates the RBE10% for 40Ar ions.The in silico and in vitro data for human laryngeal squamous cell carcinoma (SQ20B cell line) are compared in 4He, 12C, 20Ne, and 28Si ions) and 40Ar, 56Fe, and 238U ions). In case of 4He ions, the MCF MKM seems to accurately reproduce the in vitro data. By contrast, the LEM IV appears to overestimate the α and RBE10% in vitro data for 4He ions. For ions from 12C to 56Fe, the in vitro data are characterized by large values of ß  which are underestimated by both models. Therefore, due to the previously discussed anti-correlation between α and ß [10% values computed with the MCF MKM are in satisfactory agreement with the in vitro results for these ions (12C to 56Fe). Interestingly, the α values calculated by the LEM IV for ions from 12C to 56Fe agree reasonably well with the in vitro data. However, the RBE10% predicted by the LEM IV are systematically smaller than the in vitro data (12C to 56Fe ions). In case of 238U ions, the results of both models are in reasonable agreement with the in vitro data.The results of human kidney cells (T1 cell line) are plotted in  α and ß ,45, it w10% results of the LEM IV are systematically lower than the corresponding in vitro ones. The spread in the in vitro ß data prevents from drawing any conclusion on which model better describes this quantity for the TK1 cell line. As can be seen in 10% values calculated by both models are similar and in good agreement with the in vitro data. ref (=0.031 Gy−1), an unusually low αref/ßref ratio (=0.56 Gy), and relatively large in vitro values of α for the 12C ions exposures (>0.4 Gy−1). Therefore, the in vitro RBE for low dose exposures (RBEα = α/αref) shows very large values  are compared to the carbon-exposed in vitro data in The linear-quadratic model  ,18) was S is the surviving fraction as a function of the absorbed dose D, α and ß are exposure- and cell- specific fitting parameters.S (RBES) was calculated using Equation (2) [ß, αref and ßref are the linear and quadratic terms of the LQM for the radiation under investigation and the reference photon exposure respectively.The RBE for the surviving fraction tion (2) (2)RBES=All in vitro clonogenic survival data were extracted from the Particle Irradiation Data Ensemble (PIDE ) versionThe following physical information are listed for each entry: particle type, energy, LET in water, beam type (monoenergetic or a spread out Bragg peak), cell line details , and the cell cycle phase. Two sets of linear-quadratic terms are included in PIDE: the result of the fit performed by the authors of the original publication and by the PIDE team. When possible, the second set of LQM terms was preferred due to the systematic fit procedure by the PIDE team ,35). Uncref and ßref values. Similarly, we included PIDE entries for which at least one of the two LQM sets has a non-negative value of ß for the ion exposure. These negative ß values are supposed to be due to the presence of subpopulations of cells with different radiation resistance [ß. This initial filtering of PIDE led to a database containing 629 entries: 296 for human cell lines and 333 for rodent cell lines. For consistency with the in silico calculations performed with monoenergetics ions, we discarded PIDE entries for cell exposures within spread out Bragg peaks. To avoid partial cell irradiations and the large uncertainties associated with very-low energy exposures, we discarded entries in case of ions with energy <1 MeV/n. Afterwards, since this work deals with asynchronized repair-competent cell lines only, we included only asynchronized PIDE entries for which at least one of the two LQM sets  includes positive αsistance . HoweverThe following paragraphs describe the process of in vitro data selection for both rodent and human cell lines. An overview on the cell lines included in this study  is given in After the initial filtering, 333 PIDE entries were available for rodent cell lines. Since the results of Chinese hamster lung fibroblasts (V79 cell line) were topic of a previous investigation [40Ar and heavier ions) with energy less than 10 MeV/n. However, in case of ions heavier than 20Ne, it was only possible to accurately compute the microdosimetric spectra for ions of energy equal or greater than 10 MeV/n (see 85 of the remaining 122 PIDE entries were in case of repair-competent Chinese hamster ovary cells (CHO and CHO-K1 cell lines), Chinese hamster peritoneal fibroblasts (B14FAF28 cell line), and mouse embryo fibroblasts (C3H10T1/2 cell line). All the B14FAF28 entries in PIDE were extracted from a single study  and are eV/n see . TherefoThe remaining 37 rodent entries in PIDE were for 16 different cell lines. Cell lines with less than 3 entries for at least one ion were discarded. The remaining 14 PIDE entries were in case of rat intestinal epithelial fibroblasts (IEC-6 cell line), rat prostatic adenocarcinoma epithelial cells (R-3327-AT-1 cell line), epidermal cells of a newborn mice transformed with the carcinogenic dimethylbenzanthracene (PDV cell line), and squamous skin cancer cells obtained by injecting PDV cells in mice (PDV C57 cell line). Since the number of chromosomes and the radiosensitivity of the IEC-6 cell line were reported to significantly vary with the passage number , this ceref/ßref ratio of ~0.6 Gy, and the availability of morphologic and karyotypic information in the literature. The 8 cell lines with most entries in PIDE were: kidney cells (T1 cell line), skin fibroblasts (NB1RGB cell line), laryngeal squamous cell carcinoma (SQ20B cell line), myeloid leukemia cells (TK1 cell line), leukemia cells (HL-60 cell line), fetal lung fibroblasts (HF19 cell line), mammary epithelial cells (M/10 cell line), and glioblastoma cells (U-87 cell line). Entries for U-87 MG cell line were not pooled together with the ones for the U-87 cell line because of their different radiosensitivity , glioblastoma cells (A-172 cell line), and foreskin fibroblasts (AG01522 cell line) were excluded since part of other MCF MKM studies  are calculated using Equations (3) and (4) respectively.The MCF MKM  was usedc(y) accounts for the non-Poisson distribution of lethal lesions at high LET and is calculated with Equation (5).The correction factor d(y) is the dose probability density of the lineal energy y, α0 and ß0 are the LQM terms in the limit of y → 0, rd is the mean radius of the subnuclear domains, and ρ is the density (=1 g/cm3). ß  can be computed using a quadratic implementation (Equation (4)) of the correction factor c(y) (Equation (5)) used for the calculation of α [The equation used to assess α (Equation (3)) is an alternative implementation of the non-Poisson MKM . While tion of α .Rn), the mean radius of the spherical subnuclear domains (rd), the LQM terms for the reference photon exposure (αref and ßref), and the LQM terms in the limit of y → 0 (α0 and ß0). No in vitro ion-exposure data were used at any point for the assessment of these parameters. All the model parameters are representative of population-mean characteristics [Rn, rd, αref and ßref used in the MCF MKM calculations are listed in In addition to the simulated microdosimetric spectra , the MCFeristics  and are rd and, in some cases, also of Rn: the mean DNA content of the irradiated population p is the ploidy number, and ξ is a factor accounting for the cell cycle distribution of the irradiated population , p is ted cells ).p is equal to 2 for healthy cell lines and for diploid cancer cells. Though mutated chromosomes might contain an abnormal amount of DNA, an approximated value of p for aneuploid cancer cells can be calculated with Equation (7)x is the mean number of chromosomes in the aneuploid cell population and xn is the number of chromosomes in a normal set .p was set to 2 for the healthy cell lines  and for the myeloid leukemia cells (TK1 cell line) which were reported to be pseudodiploid [x was extracted from literature and it is equal to: 78 for the HeLa cell line [In this work, the ploidy number odiploid . For theell line , 44 for ell line , 62 for ell line , 59 for ell line , 39.4 foell line , and 54.ell line . Since well line .Rn. Details of the morphological information from literature can be found in Three approaches are used for the assessment of the mean radius of the cell nucleus (1) Preferably, the radius of the cell nucleus is extracted from literature as determined by means of morphologic measurements for cells presenting a spherical nucleus . This is the approach used in our previous work  dealing However, since this information is not always available, two additional strategies are used in this article. Rn) can be assessed from the cross-sectional area of the nucleus measured in case of attached cells. Generally, Rn is calculated with Equation (8)A is the cross-sectional area of the nucleus. This approach implicitly assumes that the half-thickness of the nucleus is equal to the radius assessed with Equation (8). However, the nucleus of cells attached during the microscopic measurements is not spherical, but more similar to an oblate spheroid/ellipsoid. Published results indicate that the vertical semi-axis of the oblate spheroid/ellipsoid is roughly a third of the mean radius of the cross-sectional area of the nucleus [(2) The mean radius of the spherical nucleus  and the minor semi-axis equal to a third of the equatorial radiusA is the cross-sectional area of the nucleus.Thus, we introduce an alternative formula (Equation (9)) to calculate Γ and the mean radius of the cell nucleus Rn for asynchronized cells. In this regard, Rn plotted as a function of the mean DNA content of 22 different asynchronized cell lines  If also no cross-sectional area measurements are available, we propose a novel phenomenological correlation between the mean DNA content of an asynchronized population cal data ,67,68,69The subnuclear domains in MKMs are meant to represent the dimension of subnuclear structures where the accumulation of radiation-induce damage (likely DNA DSBs) is thought to correlate with cell death ,26,70. Prd) can be assessed with Equation (11) [Therefore, under the simplifying assumption that the DNA is homogenously distributed within the cell nucleus, we hypothesize that the MCF MKM subnuclear domains represent the average volume of the cell nucleus containing 2 Mbp of DNA. Thus, the mean radius of the subnuclear domains (ion (11) :(11)rd=Rref and ßref are simply the α and ß values for that reference photon survival curve. Alternatively, as done in previous studies [ref and ßref were calculated with Equations (12) and (13):ref,i and ßref,i are the LQM terms for a reference photon exposure, and ni is the number of ion-exposure entries for that set of αref,i and ßref,i. This weighted sum was used to prevent photon experiments with few ion-exposure entries from biasing the calculation of the average αref and ßref. This approach was used for half of the cell lines included in this article, namely: CHO and CHO-K1, C3H10T1/2, HF19, NB1RGB, SQ20B, T1, and TK1.If all PIDE entries for a specific cell line share the same photon reference irradiation, then α studies ,33, in s0 and ß0 are determined by analyzing only photon in vitro data. Since the integral in Equation (5) is equal to ~1 for the reference photons, then ß0 = ßref. α0 can be calculated using Equation (14) [Similarly, αion (14) :(14)α0=αThe dose-mean lineal energy 60Co and 137Cs) and energetic X-rays (6 MV), 4.3 keV/µm for less energetic X-rays  [Since it is unpractical nor feasible to perform experiment-specific calculations of  X-rays) .1H, 4He, 7Li, 12C, 16O, 20Ne, 28Si, 40Ar, 56Fe, 238U ions were simulated. The energy of the ions was equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 MeV/n. The lineal energy spectra for ions heavier than 20Ne with energy lower 10 MeV/n were disregarded because of the presence of an inappropriate peak . A logarithmic binning from 10−2 to 107 keV/µm with 50 bins per decade was used to score the lineal energy spectra. The minimum energy deposition considered in the microdosimetric calculations was that for one event of one ionization only (10.9 eV [The computation of the microdosimetric spectra and the LET follows the methodology of our previous study with the MCF MKM  and only(10.9 eV ). http://web-docs.gsi.de/∼weick/atima/ (accessed on 5 September 2022) implemented in PHITS for the beams used to assess the microdosimetric spectra. Since the monoenergetic simulations were performed over an infinitesimal target, the simulated track- and dose- mean LET values were equivalent. By contrast, the in vitro experimental exposures are performed with quasi-monoenergetic beams. Consequently, track- and dose- mean LET values could differ. The inclusion of secondary fragments in the mean LET calculation and the use of different methods  significantly influence the LET results. Since details on the LET calculations were not always included in the publications from which the in vitro data were extracted and because different articles used different methodologies to calculate and average the LET, all results of this study are plotted as a function of a generic “unrestricted LET in water”.In order to facilitate the comparison between the in silico and in vitro data, the unrestricted LET in water was assessed using the ATIMA model  suggests that the MCF MKM can accurately predict the in vitro clonogenic survival without requiring ion-irradiated in vitro data as input. This was possible thanks to several new strategies introduced to a priori determine the MCF MKM parameters by means of morphologic and karyotypic information.The systematic benchmark performed in this work  and the use of average radiosensitivity predictions for the MCF MKM , a reasonable agreement between the results of the models was found in most cases.Future work with the MCF MKM is planned to include in the model dose-rate and fractionation effects, enhanced cell radioresistance due to hypoxia, and the transition of the survival curve to a constant slope at high doses. Additionally, we will investigate the possibility to extend the MCF MKM predictions to in vivo and patient data.


Oncological diseases have currently reached an epidemic scale, especially in industrialized countries. Such a situation has prompted complex studies in medicinal chemistry focused on the research and development of novel effective anticancer drugs. In this review, the data concerning new 4-thiazolidinone-bearing hybrid molecules with potential anticancer activity reported during the period from the years 2017–2022 are summarized. The main emphasis is on the application of molecular hybridization methodologies and strategies in the design of small molecules as anticancer agents. Based on the analyzed data, it was observed that the main directions in this field are the hybridization of scaffolds, the hybrid-pharmacophore approach, and the analogue-based drug design of 4-thiazolidinone cores with early approved drugs, natural compounds, and privileged heterocyclic scaffolds. The mentioned design approaches are effective tools/sources for the generation of hit/lead compounds with anticancer activity and will be relevant to future studies. Cancer diseases are a colossal problem for humanity, and according to data from the World Health Organization (WHO), they are the major cause of death among people under 70 years . This poMolecular-hybridization approaches are important synthetic strategies in the design of drug-like small molecules with more powerful and biologically more extensive anticancer properties than the initial compounds .4-Thiazolidinone scaffolds belong to privileged structures in drug design  and glit4-Thiazolidinone scaffolds are widely used for the development of anticancer agents, and a number of recent reviews have been published dedicated to this area of drug design ,10. The A series of publications reported the successful application of molecular hybridization methods and strategies for the design of novel anticancer agents based on obtaining hybrids of the type “approved drugs-4-thiazolidinone scaffolds”. The drugs from different pharmacological groups were used as parent molecules for further hybridization and development.1–3, were identified. Compounds 1–3 induced apoptotic cell death. Moreover, cycle arrest in the G0/G1 phase was caused by compounds 2 and 3, while compound 1 inhibited the cell cycle in the G2/M phase. Hybrid molecules 1 and 3 proved to have stronger genotoxic effects than imatinib against K562 cells [Türe et al.  reported62 cells .Recent decades have been marked by an increased interest in the use of potential NSAIDs for the treatment of cancer or using NSAIDs scaffolds/molecules for the design of new antimitotic agents . 4 and 5, were found to be efficient in some types of cancer compared to the effect of doxorubicin (reference drug), with the highest activity level detected on the MCF7 line, with IC50 values of 0.31 and 0.30 µM, respectively. Furthermore, tumor specificity and minimal effects on normal fibroblasts were observed for 4 and 5. In vitro studies of the molecular mechanisms of action for 4 and 5 revealed the induction of DNA damage, cell cycle arrest in the G2/M phase, and the induction of apoptosis as indicated by annexin-V staining and the activation of caspases. Moreover, it was found that both compounds modulate the expression and activity of several factors in the DNA damage response pathway, cyclins/cyclin-dependent kinases, and CDC25 phosphatase.Ramadan W.S. et al.  used a h6 and 7  inhibitors by Eldehna et al. . The new9  was foun10 -2-chloro-3-(4-nitrophenyl)prop-2-enal (CAS 3626-97-9) and was used as a drug in medical practice in the former Soviet Union , and LC3B (the microtubule-associated protein 1A/1B light chain 3B), suggesting its influence on the autophagy process.The diversity-oriented approach was applied for the synthesis of ciminalum-4-thiazolidinone hybrid molecules, and a series of derivatives were reported by Buzun and Finiuk ,25,26. F11  was cyto12 [50 = 1.57 µM and TGI = 13.3 µM). The analysis of activity inside cancer panels revealed a certain sensitivity profile in the GI50 concentration range of < 0.01–0.02 µM toward leukemia , colon cancer (SW-620), CNS cancer (SF-539), and melanoma (SK-MEL-5) cell lines. In the same paper [13  and caused apoptosis toward human T-leukemia cells of the Jurkat line. However, the application of 14 and 15 did not affect the morphology and the DNA intactness in mitogen-activated lymphocytes of the peripheral blood of healthy human donors.Finiuk et al.  reported4 and 15 , possessThe structural motifs of natural products always serve as an excellent source for drug-like molecules from the perspective of the design of anticancer agents ,28.16 and 17  and lung cancer cell line A549 (IC50 = 1.28 ± 0.98 μM). The cell cycle analysis disclosed that 25 showed significant G2/M phase arrest in MDA-MB-231 cells. Furthermore, hybrid 25 significantly inhibited tubulin polymerization, with an IC50 value of 3.54 ± 0.2 µM, and caused apoptosis-mediated cell death in MDA-MB-231 cells. In silico studies inferred that hybrid 25 binds at the colchicine binding site of the tubulin, with prominent binding affinity.The synthesis and evaluation of antiproliferative activity in A549 (lung cancer), MDA-MB-231 and BT-474 (breast cancer), HepG2 (liver cancer), and HCT-116 (colon cancer) cell lines using an MTT of coumarin-2–iminothiazolidin-4-one hybrids were reported by Sigalapalli et al. . Hybrid 25  showed eSteroids are important and physiologically significant natural compounds, opening up the possibility of structural tuning with the aim of achieving and developing valuable pharmacological properties including potent anticancer activity . Živković et al.  designed50 of the majority oscillated around 10 µM, with the IC50 of the cisplatin reference compound for these lines being 5.7 µM and 5.2 µM, respectively. Compared to the positive control, these compounds exhibited lower toxicity to normal cells. Hybrids 26 and 27 were found to be the most active . Furthermore, molecule 34 exhibited superior protection of normal HDF cells than 33. Additionally, the anticancer potency of the compounds was justified by using in silico studies. SAR analysis concluded by the authors suggests the importance of the presence of dichlorosubstituted arylamino moiety in position two of the 4-thiazolidinone core in enhancing potency.A series of 4-thiazolidinone-pyrazole hybrids connected via a ylidene linker were synthesized by Bhat et al. . An in v3 and 34  exhibite35 -3-(5-methylisoxazol-3-yl)thiazolidin-4-one, 42 , as a po42 exhibited excellent anticancer activity against MCF7 and displayed less cytotoxicity towards normal HEK293 cell lines. Hybrid molecule 42 inhibited the cell proliferation of HeLa by 58%, 87%, and 94% at 6.25 µM, 25 µM, and 50 µM concentrations, respectively. In terms of inhibition, 59% and 65% inhibition were observed at a 50 µM concentration on breast cancer (MCF7) and lung cancer (A549) cell lines, respectively, by compound 42. At lower concentrations, compound 42 has less effect on normal cell lines (HEK293). Additionally, a molecular docking study explored the binding mode and possible interactions between the synthesized compound and the ATP binding site of the EGFR kinase domain.The results of an in vitro cytotoxicity assessment employing the MTT assay as per the ATCC protocol, using three human cancer cell lines, HeLa , MCF-7 (breast cancer), A549 (human lung cancer), and HEK-293 , indicated that hybrid 43 (43 demonstrated inhibition activity against (GI50 < 10 μM) against all 59 human tumor cell lines, with average GI50/TGI/LC50 values of 2.57/57.27/94.71 μM, respectively. A series of 5-enamine–4-thiazolidinones with phenylalanine moiety in the molecules was designed and reported by Holota et al. . Among t43 , with a Preliminary SAR provided by the authors revealed that the presence of thiazole substituent is crucial for anticancer activity whereas a change in the hydrogen atom, aniline derivatives, or 1,2,4-triazole nucleus leads to the disappearance of the effect.44 and 45 were found to be the most active against cancer cell lines, with total mean GI50 values of 3.54 µM (44) and 10.96 µM (45), without causing toxicity to normal somatic HEK293 cells, with IC50 values of 28.99 µM (44) and 24.43 µM (45).Two series of merged 1,2,4-triazole–4-thiazolidinone hybrids with ylidene and enamine linkers in the molecules  were desAn SAR analysis performed by authors revealed that both the presence and position of a chlorine atom in the benzylidene area of molecules play a crucial role in the realization of the anticancer effect .46 and 47  and HepG-2 (liver cancer cell line) were IC50 values of 13.0 µM and 19.2 µM, respectively, for hybrid 46 and values of 12.4 µM and 16.2 µM, respectively, for 47.A series of 17 novel pyridine-thiazolidinone hybrids were synthesized, characterized, and evaluated as potential hCA inhibitors by Ansari et al. . Of all 6 and 47  had the 48 and 49  production, and modulated redox status.The molecular hybridization approach was applied for the synthesis of pyridine–4-thiazolidinone hybrids with aim of the synthesis of potential antiglioblastoma agents by Campos et al. . Among s50–52  displaye53 .The attempt to design potential antimitotic agents following the hybridization of pyridine, piperazine, and 4-thiazolidinone scaffolds was reported by Demirci et al. . The syn53  showed t54 , the IC50 value for the compound was 10.84 µM, indicating that compound 55 is 10-fold more selective against the colon cell line. SARs analysis performed by the authors revealed that the antiproliferative activity of phenethyl-thiazolidinone-indole hybrids is higher than that of benzyl-thiazolidinone-indole ones. Furthermore, it was observed that hybrids without a substituent at the nitrogen atom into indole core are more active than N-alkyl/aryl-substituted ones. In addition, the presence of the methoxy group at the phenyl ring of the side chain of hybrid compounds leads to higher activity than halogen substituents. Extended analyses on compound 55 revealed that the compound inhibited the cell cycle of HCT-15 cells in sub G1 and G2/M phases, additionally inhibited tubulin more strongly compared to the reference compound, induced intracellular ROS, and decreased the mitochondrial potential, causing cell death by apoptosis [Indole scaffolds are often used for the design and development of 4-thiazolidinone hybrids as anticancer agents. Sigalapalli et al.  reported55  showed tpoptosis .56  possessed moderate sensitivity to hybrid 57. Furthermore, compound 57 induced apoptosis in studied tumor cells via caspase-3-, PARP1-, and Bax-dependent mechanisms; however, it did not affect the G1/S transition in HepG2 cells. Additionally, hybrid 57 impaired nuclear DNA in HepG2, HCT116, and MCF-7 cells without intercalating this biomolecule, but much fewer DNA damage events were induced by 57 in normal Balb/c 3T3 fibroblasts compared with HepG2 carcinoma cells.A series of novel indole-azolidinone hybrids with 5-fluoro-3-formyl-1reaction . Among ttion [57  was foun®) [Isatin  is an en®)  was esta58  and MCF-10A , with IC50 values of 49.1 µM and 73.1 µM, respectively. In addition, hybrid 58 significantly induced the expression of the pro-apoptotic protein Bax, inhibited the expression of the anti-apoptotic protein Bcl-2, and increased the level of caspase-3 compared to the control sample.El-Naggar et al.  reported58  possesse59  activity. In addition, in silico and in vivo studies demonstrated that compound 59 has an excellent physicochemical and pharmacokinetic profile (which PK and docking can develop).Fouad et al.  develope59  was effe60  cell line was evaluated. The results obtained by the authors showed that the studied molecule 60 exhibits both cytotoxic and proapoptotic properties at a 10–100 µM concentration range. The activation of caspase-3 was caused by 60 and was accompanied by an LDH release and resazurin reduction. Additionally, increased DCF fluorescence in a wide range of concentrations after 6, 24, and 48 h of exposure was observed after treatment with 60, which suggests the stimulation of ROS production. The properties of 60 established by the authors make it a promising tool for further research as an anti-cancer agent.Szychowski et al.  reported60 . The imp61 , with the preservation of biochemical and histochemical structures being close to normal.Nafie et al.  studied 62  was the 63  that hybrid 63 arrested the cell cycle at the G2/M phase and possesses binding properties with N-acetyl transferase (hNAT-1) protein. The authors suggest that the identified quinolinone–4-thiazolidinone hybrid 63 could be considered as a new chemotype targeting hNAT-1 and could be used for hit/lead compound generation in anticancer drug design and discovery.A new series of quinolone–4-thiazolidinone hybrid molecules were reported by Kumar et al. . All the63  was founQi et al. ,65,66,6764,65,68) and toward HT-29 (human colon cancer cell line) (66–68) were identified.Structural modification of phenyl rings A and B as well as quinoline ring C was applied  in the mm-position in the phenyl ring attached to the 4-thiazolidinone nucleus at position two (69) was found to be the most active against the hepatic cell line, with an IC50 of 1.79 µg/mL. A series of hybrids with a quinazoline scaffold was synthesized by Samridhi Thakral et al.  Figure . The synp-position in the mentioned phenyl ring (70) leads to activity against breast cancer cell line MCF-7, with an IC50 of 1.94 µg/mL. The IC50 value for the standard drug in this study was found to be 0.09 µg/ml.The presence of the methoxy-group at the 50 values from 0.096 µM to 0.98 µM) on lung cancer (A549) cell lines, and four compounds showed a broad spectrum of cytotoxic activity on all the examined cancer cells in the range of 0.096–4.58 µM. Among them, compounds 71 and 72  of 0.096–0.32 µM and with low cytotoxicity in the HeK-293T line, with IC50 values of 6.76 and 6.65 µM respectively.Sharma et al.  reported1 and 72  were fou71 and 72 on A549 lung cancer cells revealed a remarkable inhibition of cell migration through the disruption of the F-actin assembly. Moreover, treatment with 71 and 72 led to the collapse of the mitochondrial membrane potential (DJm) and increased the levels of ROS in A549 cells. Reported in [In-depth studies of the impact of orted in  were theIqbal et al.  reported73–75  and extrinsic pathways, with the upregulation of anti-apoptotic signals and downregulation of pro-apoptotic signals, were the most often indicated. An important reported mechanism for antimitotic activity was an impact on the cell cycle with arrest following in different phases. Moreover, enzymes such as PIM-and tyrosine kinases as well as carbonic anhydrases were used as molecular targets for the design of novel 4-thiazolidinones with anticancer activity. Some reported hybrids were found to be promising tubuline inhibitors and inducers of ROS generation. The molecular hybridization strategy is a popular trend and an attractive research direction, providing endless source of significant opportunities in the design of new 4-thiazolidinone-bearing hybrid molecules with potential anticancer activity. A combination of potential pharmacophores in one molecule is one of the possible approaches used to achieve multi-target and polypharmacological effects for small molecules and is especially essential in the case of antimitotic agents. The hybridization of scaffolds, the hybrid-pharmacophore approach, and analogue-based drug design are key tools that are widely used by synthetic and medicinal chemists in this field. The hybridization of the 4-thiazolidinone core with early approved drugs, natural compounds, and privileged heterocyclic scaffolds is the most frequently used method in the development of novel molecules with antimitotic effects. The application of a molecular hybridization methodology allowed for identification of the new “hit” and “lead” hybrid compounds which target valid and important targets in carcinogenesis. The mentioned strategies and methodologies will remain relevant to studies over the next decade, and new wave/direction in the design of potential antimitotic agents using the hybridization of 4-thiazolidione scaffolds with biomolecules such as monoclonal antibodies, etc., can be expected. The data presented in this review contribute to the SAR profile of 4-thiazolidinone-based hybrid molecules for further exploration in the design, improvement, and optimization of new molecules with antitumor activity.


The plasma membrane separates the interior of the cells from the extracellular fluid and protects the cell from disruptive external factors. Therefore, the self-repairing capability of the membrane is crucial for cells to maintain homeostasis and survive in a hostile environment. Here, we found that micron-sized membrane pores induced by cylindrical atomic force microscope probe puncture resealed significantly (~1.3–1.5 times) faster in drug-resistant non-small cell lung cancer (NSCLC) cell lines than in their drug-sensitive counterparts. Interestingly, we found that such enhanced membrane repairing ability was due to the overexpression of annexin in drug-resistant NSCLC cells. In addition, a further ~50% reduction in membrane resealing time  was observed through the epithelial-mesenchymal-transition, highlighting the superior viability and potential of highly aggressive tumor cells using membrane resealing as an indicator for assessing the drug-resistivity and pathological state of cancer. In addition to serving as the physical barrier separating cytoplasm from outside, and protecting the cell from disruptive extracellular factors, the plasma membrane also allows a cell to achieve signal and mass exchange with the surrounding microenvironment  and maintain homeostasis is critical for the cell’s survival. For example, it is widely reported that cells must actively pump out calcium ions to keep their internal concentration low, a condition that is critical for the proper functioning of many proteins and signaling pathways . HoweverMicroscopically, it is well-documented that the calcium influx through the ruptured membrane could trigger a cascade of events to help its repairing. In particular, annexin family proteins  were used in this study. The HCC4006 cell line was cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS), and ETCC016 cells were cultured in DMEM supplemented with 10% FBS and 1% PS. Both cell lines were maintained in a 37 °C humidified incubator containing 5% COBoth HCC4006 and ETCC016 cell lines were treated with 5 μM erlotinib for over 10 passages to gain resistance to the drug. We named the resistant phenotype of these cell lines as HCC4006R and ETCC016R, respectively, whereas the parental control groups  were treated with the same amount of dimethyl sulfoxide (DMSO), instead of erlotinib.To evaluate the IC50 value of erlotinib in both drug-sensitive and drug-resistant cell lines, MTS assay CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit  was performed. Briefly, 0.0001 µM, 0.001 µM, 0.01 µM, 0.1 µM, 1 µM, 10 µM, 20 µM, 50 µM, 100 µM, 200 µM erlotinib and control (DMSO) were added to both cell lines. After 72 h, the supernatant was discarded and replaced with MTS reagent solution (20 µL/well for 96-wellplate), and cells were then incubated for another 4 h. The absorbance of 490 nm wavelength was read by a plate-reader to measure the viability of cells.EMT in both HCC4006 and ETCC016 cell lines was activated through TGF-β receptor signaling pathways. Specifically, 4 ng/mL TGF-beta was added to the growth medium 36 h before experiment . The occCells were digested and transferred into glass-bottom dish  precoated with poly-L-lysine , tested after 30 min incubation to ensure cells would adhere strongly to the dish. Membrane puncturing experiment was conducted using atomic force microscopy (AFM). Specifically, cylindrical AFM probes (with a diameter of 2 µm) were fabricated by focused ion beam (FEI Quanta 200 3D). After that, said probe was pushed into the cell to induce membrane damage (similar to that achieved by using laser or electric pulses ). The recP) and external (eP) pressures were balanced by the membrane tension γ m via the Laplace’s law asR represents the cell radius. Since the indentation depth is small, the contact area A and indentation depth δ can be related to each other as:The membrane tension of cells was estimated by indenting the cell membrane with a cylindric AFM, a method that has been used in previous studies ,20. BefoF applied on membrane through the cross-section area A of the indenter must equal to that induced by the pressure difference across the membrane, i.e.,On the other hand, the force Combining the above equations, the membrane tension can be estimated as:Cells were treated with cold methanol for 10 min to make their membrane permeable to dyes (without destroying embedded membrane proteins), followed by another 10 min treatment of PBS with 0.25% Tween 20. After being washed with PBS for 3 times, the samples were immersed in 5% BSA blocking solution for incubating 30 min at room temperature. Next, primary monoclonal antibody annexin A2  diluted in 2% BSA PBS and added. After overnight incubation at 4 °C and washing with PBS 3 times, a secondary antibody was added and the samples were incubated at room temperature for 1 hr. DAPI  was added into the medium 30 min before observation.The fluorescence images were obtained using Nikon Eclipse Ts2R-FL fluorescence microscope with fixed exposure time for each sample.To evaluate the expression of EMT markers, real-time quantitative PCR (RT-qPCR) was employed to detect the expression levels of E-cadherin, N-cadherin and vimentin. To further determine the role of proteins in relation to membrane repairing capability, annexin A2, A5, A6, A7 were also tested. GAPDH was determined as the reference gene. The primers were purchased from Lifeact Technology . TRIzol Reagent  was used to lyse the cell to extract mRNA, following the manufacturer’s instructions. RT-qPCR was performed with a QuantiNova SYBR Green PCR Kit . Specifically, the cycling program was adopted as: (i) denaturation at 95 °C for 15 s, (ii) annealing at 60 °C for 30 s, and (iii) repeating these two steps for 45 cycles .Using an atomic force microscope (AFM) mounted with a 2 μm cylindrical probe refer to a, membraWith the method for introducing membrane pores with precisely controlled size at hand, we can then examine how the self-repairing capability of cancer cells is influenced by their pathological status. Given its important clinical implications, we first investigated how the drug resistance developed in non-small cell lung cancer cells affect their membrane resealing ability. Specifically, drug-resistant ETCC016 and HCC4006 cells were developed by culturing these two cell lines with 5 μM erlotinib for more than 10 passages. The resistance to erlotinib of these cell lines was evaluated/verified by the MTS assay shown in As pointed out earlier, membrane tensions in these drug-resistant and drug-sensitive cells were found to be more or less the same, therefore we look for other factors that could give arise to the different membrane resealing response observed here. In particular, we examined the expression of annexin in these cell lines, given the well-known role of such proteins in facilitating membrane remodeling. As shown in Another way for tumor cells to acquire drug-resistance is the EMT, where epithelial-like cells lose their apical-basal polarity and adhesion with each other, and eventually become highly migratory. Specifically, the reduced drug-induced apoptosis and elevated drug efflux pumping ,25 in tuWe proceeded by adding 4 ng/mL TGF-β, a common EMT inducer, into the culture medium of cells. Interestingly, as shown in It must be pointed out that, among all cell lines tested, HCC4006R underwent the largest increase in the annexin expression level (after TGF-β treatment) coupled with the biggest decrease of membrane resealing time (from ~23 to ~13 s) which again supports the notation that EMT enhances the membrane repairing capability of cells. Similar to the previous drug-sensitive and drug-resistant case, we then examined how TGF-β treatment influences annexin expression in NSCLC cells. As expected, a much bigger increase in the expression level of annexin A2, A5, A6 and A7 was observed in drug-resistant HCC4006R cells, in comparison to the HCC4006M cell line a. On theThis study showed that micron-sized membrane pores (induced by AFM puncturing) in NSCLC cells could fully reseal in tens of seconds. Interestingly, although membrane tension remained more or less the same across cancer cell lines, erlotinib-resistant tumor cells were found to reseal their membranes much faster than the corresponding drug-sensitive cell lines. We further demonstrated that such enhanced membrane repairing capability was due to the overexpression of annexin (a protein family known to play significant roles in the remodeling and reorganization of lipid membranes) in drug-resistant NSCLC cells. Interestingly, EMT could further reduce the resealing time of the membrane. These findings are consistent with previous studies reporting that annexin facilitates membrane repairing by inducing curvature and folding of lipid bilayer near the pore edge , and knoIt must be pointed out that clinically, overexpression of annexin A2 in the tumor was considered a predictor of adverse outcomes for patients . Recent Finally, given that membrane puncturing and resealing tests can be conducted in a few minutes, it is conceivable that we might be able to use membrane resealing time as a marker for rapid drug-resistance identification and/or classification of cancer. Indeed, cell stiffness level has been successfully applied to distinguish tumor cells from their normal counterparts . Finally


Carcinomas are characterized by a widespread upregulation of intercellular junctions that create a barrier to immune response and drug therapy. Desmoglein 2 (DSG2) represents such a junction protein and serves as one adenovirus receptor. Importantly, the interaction between human adenovirus type 3 (Ad3) and DSG2 leads to the shedding of the binding domain followed by a decrease in the junction protein expression and transient tight junction opening. Junction opener 4 (JO-4), a small recombinant protein derived from the Ad3 fiber knob, was previously developed with a higher affinity to DSG2. JO-4 protein has been proven to enhance the effects of antibody therapy and chemotherapy and is now considered for clinical trials. However, the effect of the JO4 mutation in the context of a virus remains insufficiently studied. Therefore, we introduced the JO4 mutation to various adenoviral vectors to explore their infection properties. In the current experimental settings and investigated cell lines, the JO4-containing vectors showed no enhanced transduction compared with their parental vectors in DSG2-high cell lines. Moreover, in DSG2-low cell lines, the JO4 vectors presented a rather weakened effect. Interestingly, DSG2-negative cell line MIA PaCa-2 even showed resistance to JO4 vector infection, possibly due to the negative effect of JO4 mutation on the usage of another Ad3 receptor: CD46. Together, our observations suggest that the JO4 vectors may have an advantage to prevent CD46-mediated sequestration, thereby achieving DSG2-specific transduction. Carcinomas, malignancies of epithelial tissue, represent the majority of all human cancer cases . The key6 PtDd per infectious virus), and it has been hypothesized that PtDd contributes to virus escape and spread [Adenoviruses have evolved mechanisms to breach epithelial barriers . For exad spread .To address the issue of the reduced carcinoma penetration rates of systemically applied oncotherapy, the high-affinity junction opener 4 (JO-4) was developed by mutagenic libraries . JO-4 prMacaca fascicularis [The binding of JO-4 protein to DSG2 leads to the shedding of DSG2 followed by a decrease in junction protein expression and transient tight junction opening. JO-4 protein’s synergy with antibody and chemotherapy was proven in efficacy studies with the chemotherapy drug Doxil  and ovarian cancer in a mouse xenograft model . Furthericularis . Currenticularis ,27. Receicularis . This HDPreviously, other high-affinity fiber knobs from Ad35 were explored to improve the in vitro and in vivo infection properties . For theOur study aimed at producing high-DSG2-affinity viral vectors to improve cell uptake and tumor penetration depth. To this end, we constructed three novel vectors containing the JO4 mutation and investigated them in a panel of tumor cell lines with different DSG2 expression levels to assess these vectors for their infectivity, transgene expression ability and oncolytic effects in direct comparison with their parental viruses. We found that the high-affinity JO4 mutation did not result in correspondingly higher transduction rates of individual viruses. However, we observed that the DSG2 level played a crucial role in the JO4-containing viruses’ activity. In the high-DSG2-expressing cell lines Hs578T and Hela, the JO4-containing viruses displayed comparable transduction levels with their individual parental viruses. In comparison, in the DSG2-negative cell line MIA PaCa-2, the JO4 vectors showed significantly weaker effects than their parental vectors. We further analyzed the effect of JO4 on the usage of CD46 and CAR, another two major adenovirus receptors. CD46 is proven to be a binding and entry receptor for Ad3, especially in the absence of DSG2 ,34. HereThe JO4 mutation was introduced into the adenoviral genome containing plasmids as previously described ,36,37. ITo rescue these vectors, the plasmid backbone was removed with pre-inserted restriction enzymes; the released adenovirus genome containing the aimed for modification was transfected into HEK293 cells with calcium–phosphate transfection reagents. After serial passaging to amplify the vectors to large scale, the vectors were purified in two rounds of cesium chloride (CsCl) gradient, as previously described . The ade2.Cell lines Hela, HEK293, Hs 578T, HNC, Huh 7, MDA-MB-231, CHO, Caco-2, MIA PaCa-2 and A549 were cultured in high-glucose Dulbecco’s modified Eagle’s medium . CHO cells were further supplemented with 1% non-essential amino acid solution . T84 was supplied with half DMEM, half Ham’s F12 (PAN-Biotech). Jurkat and K562 cells were cultured in RPMI 1640 (PAN-Biotech). All the above media were supplemented with 100 units per mL penicillin and 100 µg per mL streptomycin (PAN-Biotech). The Caco-2 medium was supplemented with 20% FBS, while all other media contained 10% FBS from the same supplier (PAN-Biotech). All cells were maintained in a humidified atmosphere at 37 °C and 5% COFor the transwell culture experiments, T84 cells were seeded into collagen-coated transwells . For this purpose, cells were counted, and 80,000 cells were seeded per well. Cells were cultured in T84 medium see  from apiTo form spheroids, approximately 1000 cells per well were seeded of different carcinoma cell lines into U-bottom 96-well plates  in their respective media. Brightfield and fluorescence images were taken every 12 h using a Satorius Incucyte SX5 live cell imaging system. Numeric evaluation was performed with the Incucyte spheroid software .5 cells were seeded in 24-well plates. After achieving a complete confluence, cells were infected at various viral particles per cell (vp/c) and incubated overnight. After 24 h, cells were washed once with Dulbecco’s Phosphate Buffered Saline (DPBS) and detached with trypsin-EDTA. Cells were resuspended in DMEM containing 10% FBS, followed by a centrifugation step , and washed in DPBS before fixation in 2% paraformaldehyde (PFA). Fluorescence profiles were obtained by analyzing at least 10,000 viable cells on a CytoFlex flow cytometer . A background signal was obtained by analyzing the negative control consisting of uninfected cells. The percentage of GFP-expressing cells was determined by selecting the region of fluorescence above the background of auto-fluorescence from uninfected cells.To detect the transduction efficiencies in different tumor cell lines, cells were analyzed with flow cytometry. First, 1 × 105 cells were washed with PBS supplemented with 1% BSA, centrifuged  and resuspended in 100 μL PBS/BSA and 1 μL PE-conjugated rabbit anti-hCAR antibody . Following an incubation step at room temperature for 1 h, cells were washed again with PBS/BSA to remove unbound antibodies and resuspended in 100 μL PBS for flow cytometry (Beckman Coulter CytoFlex flow cytometer). The PE-conjugated mouse anti-human CD46 antibody  was used to detect the surface expression of CD46 on different cell lines. For the DSG2 detection, Alexa Fluor 488-conjugated mouse anti-human Desmoglein 2 antibody was used .To detect CAR (coxsackievirus and adenovirus receptor) expression on the cell surfaces of different tumor cell lines using flow cytometry, 1 × 10The TEER of the T84 cellular barrier was measured in ohm to evaluate the barrier integrity. The transwell culture, which consisted of a multiple layer of cells cultured on a semipermeable filter insert, produced a partition for the inner (or upper) and outer (or lower) chambers. For the electrical measurements, Millicell ERS-2 Voltohmmeter  was applied. Two electrodes, one placed in the inner and one in the outer chamber, were used to measure the resistance of the cellular multilayers.The histological analysis of the T84-transwell cultures was performed to visually evaluate cell differentiation and integrity. Briefly, the 3D cultures were fixed in 4.5% formaldehyde/PBS. After 24 h of fixation, the round membrane was cut out from the transwell using a sterile and sharp scalpel and padded on blue tissue foam . This was subsequently soaked in tap water for 2 h. After dehydration in alcohol, clearing in xylene and embedding in paraffin, the blocks were cut to a thickness of 5 µm with a rotary microtome . Then, the slides were incubated overnight at 37 °C. The blocks were then de-paraffinized at high temperatures, followed by xylene treatment and rehydration with ethanol and washing with deionized water. Subsequently, hematoxylin and eosin staining were performed. The sections were examined under a microscope.To quantify the cell entry efficiency, a defined number of adenovirus particles (vp) was used to infect pre-seeded cells in 24-well plates, which were then incubated for different time periods. Cell monolayers were digested and flushed off with trypsin, followed by intensive washing with PBS. Genomic DNA was extracted using Monarch Genomic DNA Purification Kit . To monitor virus genome uptake efficiency, quantitative polymerase chain reaction (qPCR) analysis was performed.To monitor virus genome uptake efficiency, duplex-qPCR was performed with primers and a probe, detecting the Ad3 fiber knob region  to quantOncolytic assays were performed in 48-well plates. A 3-fold dilution series of individual adenoviruses was freshly prepared to infect pre-seeded cancer cells. The development of any cytopathic effect (CPE) was checked daily until at least one of the viruses on one plate at the lowest dosage showed CPE. The experiment was maintained for a maximum of 14 days. The cells were first fixed with 3.7% formaldehyde, and then the maintained adherent cells were detected by staining the attached cells with 0.5% crystal violet dye. After several wash steps, the stained plates were dried and photographed.Plaque assays were performed following a previously published protocol . In deta9 vector particles was added to SDS-loading buffer, heated for 5 min at 70 °C and loaded on an 8% separation/5% stacking SDS-gel. For Ad3 and Ad3–JO4, a 4–15% gradient gel Mini-PROTEAN TGX  was used. Both gels ran at 80 V. The separated vector proteins were stained silver as previously described [An amount of 1 × 10escribed ,41.Statistical analyses were conducted with GraphPad Prism 8 . We used the nonparametric Mann–Whitney U test to identify significant differences between the JO4 vectors and their individual parental vectors .The aim of this work was to transfer the previously identified junction opener protein 4 (JO-4) into a viral vector setting. We first generated viral vectors with the JO4 mutation and then explored the effect of different JO4-containing adenoviral vectors in their infectivity, transgene expression ability and oncolytic effect in direct comparison with their individual parental vectors. To test the potential therapeutic benefits of the JO4 variant, we explored different cancer cell lines in two-dimensional (2D) and 3D culture regarding transgene expression level and virus cellular entry. Moreover, to investigate the correlation between virus infection and receptor expression, cell lines with different major adenovirus receptor expression levels were included in this study. Furthermore, these vectors were evaluated in CAR and CD46 knockout A549 cells as well as in stable CD46- and CAR-expressing CHO cells.To investigate the affinity-enhancing JO4 mutation in the context of a virus, we constructed three new vectors containing the JO4 mutation (JO4 vectors) A–C. The To confirm that all JO4 vectors are properly expressing fiber protein, the virus particles were lysated with SDS containing buffer and loaded on silver gel. As shown in To explore the effect of JO4 on virus infectivity, the JO4-containing vectors and their parental vectors were investigated in a panel of tumor cell lines. A previous study of CD46 high-affinity Ad35 fiber knob (Ad35++) revealed that the CD46 density played an essential role in the cell line transduction . TherefoFirst, we performed the experiments in monolayer cell culture. Since colon adenocarcinoma-derived Caco-2 and T84 cell lines are frequently used as in vitro models of functional epithelial barriers, we chose these two as models for analyzing the high-affinity DSG2 vectors. The two cell lines showed similar major adenovirus receptor levels (CD46 and CAR) and around 75% DSG2 expression . Here weAs many cancers with hematopoietic cell origin show strongly upregulated DSG2 expression and present a potential target for oncolytic viral therapy , we alsoThe ability to spread in a tumor is an important feature for the application of oncolytic viruses in solid tumors. Here we investigated the JO4-containing vectors in monolayer cell culture regarding plaque forming and cell lysis .We first assessed the two replication-competent viruses Ad3 and Ad3-JO4 in A549 cells and in the CD46 knockout A549 cell line (A549-CD46-KO) by applying an agarose overlay to evaluate the virus’s spreading ability. No difference was observed between the two viruses regarding plaque size or number A. It wasFurthermore, we investigated the Ad3-JO4 vector in spheroid culture, which has been used in cancer research as an intermediate model between in vitro cancer cell line cultures and in vivo xenograft tumors. We used a panel of human carcinoma cell lines that were able to grow as dense spheroids and showed high DSG2 expression (over 80% of cells expressed DSG2) including MDA-MB-231 (breast), Hela (cervix), Huh 7 (liver) and HNC (head and neck) cells A–D. An aSince the natural target of Ad3 is polarized epithelial tissue, we then studied the spread of Ad3 and Ad3-JO4 in a colon cancer T84 cell transwell culture model. T84 cells were cultured on semipermeable filter inserts in order to form a multilayer structure with intercellular junctions for four weeks. Amounts of 400 vp/c of Ad3 and Ad3-JO4 were added to the apical chamber. Trans-epithelial electrical resistance (TEER) was measured at the indicated time points E. There Since we observed a correlation of DSG2 expression level and JO4 vector behavior, we investigated JO4 vectors’ usage of CD46 and CAR. As the CHO-K1 wild-type cell line expresses none of the three primary adenovirus receptors, CD46, CAR or DSG2, and allows neither Ad3 nor Ad3-JO4 to transduce, it is a suitable model for studying the interactions of these two vectors with CD46 or CAR exclusively, using stable CD46- or CAR-expressing CHO cell lines A. The FAFurthermore, we evaluated all three JO4 vectors in comparison with their parental vectors in wild-type A549 cell line, CD46 or CAR knockout and CD46 and CAR double- knockout A549 cell lines B–E. Ad5 Virus attachment and cell entry are the first steps of infection, and the latter is also used to define the infectious titer or transducing units. Here we quantified the virus genome inside the cells at certain time points postinfection in qPCR . In HEK Viruses can interact not only with well-displayed receptors at the apical side of epithelial cells but also with poorly accessible receptors hidden in cell to cell junctions . The molNotwithstanding its promising application as a recombinant protein, definitive proof of the function of JO4 in the viral life cycle is elusive. Therefore, we generated three adenoviral vectors with the JO4 mutation in the fiber knob. The first two groups are replication-defective chimeric vectors based on E1-deleted Ad5, with either the whole fiber region from Ad3 (F3) or only the fiber knob from Ad3 (K3). The third pair are replication-competent Ad3 vectors. The transduction was first evaluated in a panel of tumor cell lines with DSG2 levels ranging from zero to one hundred percent receptor-positive cells . SurprisIn two-dimensional (2D) submerged culture, we did not observe any difference with plaque-forming assays but saw three- to ten-fold less efficiency of Ad3-JO4 compared with Ad3 in oncolytic assays, suggesting the reduced virus transduction of these tumor cell lines . Since tBased on the distinguishing features of JO4 viruses in cancer cell lines with different DSG2 levels, we hypothesized that not only DSG2 binding but also interaction with other molecules or the lack thereof played a crucial role in the life cycle of JO4 viruses. In addition to DSG2, CD46 has proven to be a minor receptor for Ad3 infection, and CAR was also hypothesized as such ,34,47. TWe suspect that the valine in position 239 of the fiber knob amino acid sequence serves as a region crucial for Ad3 fiber knob binding to CD46. Moreover, human A549 cells and individual CD46 and CAR knockout cell lines were included. However, here the receptor knockout did not induce a difference in virus transduction between with and without JO4 B–E. We sA receptor density effect was previously observed in high-affinity Ad35 fiber knob containing viruses (Ad5/35+ and Ad5/35++) . No signCurrently, Ad5-based chimeric vectors with fiber knobs from Ad3 and pure Ad3-based vectors represent promising alternatives to traditional Ad5 vectors in terms of receptor usage and preexisting immunity ,55,56; iIn sharp contrast to the efficacy observed with the single JO-4 protein, the JO4 mutation resulted in no enhanced efficiency in the context of a virus, probably due to the negative influence of this mutation on CD46 interaction. Therefore, JO4-containing vectors should be considered in applications to compete with the CD46-mediated sequestration of vector particles in directing DSG2-specific transduction.