Menin/MEN1 is a scaffold protein that participates in proliferation, regulation of gene transcription, DNA damage repair, and signal transduction. In hematological malignancies harboring the KMT2A/MLL1 (MLLr) chromosomal rearrangements, the interaction of the oncogenic fusion protein MLLr with MEN1 has been shown to be essential. MEN1 binders inhibiting the MEN1 and KMT2A interaction have been shown to be effective against MLLr AML and B-ALL in experimental models and clinical studies. We hypothesized that in addition to the MEN1–KMT2A interaction, alternative mechanisms might be instrumental in the MEN1 dependency of leukemia. We first mined and analyzed data from publicly available gene expression databases, finding that the dependency of B-ALL cell lines on MEN1 did not correlate with the presence of MLLr. Using shRNA-mediated knockdown, we found that all tested B-ALL cell lines were sensitive to MEN1 depletion, independent of the underlying driver mutations. Most multiple myeloma cell lines that did not harbor MLLr were also sensitive to the genetic depletion of MEN1. We conclude that the oncogenic role of MEN1 is not limited to the interaction with KMT2A. Our results suggest that targeted degradation of MEN1 or the development of binders that induce global changes in the MEN1 protein structure may be more efficient than the inhibition of individual MEN1 protein interactions. HOXA genes, MEIS1 [CDK6, MEF2C [FLT3, that might have major pathogenetic significance [KMT2A in these neoplasms [Mutated in endocrine neoplasia menin/MEN1) is a scaffold protein that participates in multiple biological processes, including proliferation, regulation of gene expression, and DNA damage repair  is a sca,3. MLLr s, MEIS1 , CDK6, M6, MEF2C , and FLTificance ,7. Most ificance . In addiificance  and meniificance  confer deoplasms . Clinicaeoplasms ,12. In peoplasms , while Keoplasms .Surprisingly, a dependency on MEN1 was revealed not only in MLLr-, NPM1m-, or MN1m-driven leukemia, but also in other types of neoplasms , which were driven by alternative mutations ,16,17. GTo test this hypothesis, we analyzed publicly available data from genome-wide loss-of-function screenings. Comparing AML and B-ALL, two leukemia types often driven by MLLr, MEN1 dependency correlated with the presence of MLLr only in AML. We validated the data of CRISPR loss-of-function screenings using shRNA-dependent knockdown and found that all tested B-ALL cell lines were sensitive to MEN1 depletion, independently of the type of driver mutations. Vice versa, only cell lines driven by MLLr were sensitive to VTP50469. Most of the multiple myeloma (MM) cell lines were also sensitive to the genetic depletion of MEN1, but none of them were sensitive to the inhibitor of MEN1–KMT2A interaction VTP50469 at therapeutically relevant doses. We conclude that the role of MEN1 in leukemia is not limited to its interaction with KMT2A, hence pharmacological degradation may be a better approach than the inhibition of individual MEN1 PPIs.https://depmap.org/portal/depmap/; accessed on 24 October 2022). Thus, we used DepMap to corroborate previous data .To further understand the role of MEN1 in the oncogenic programs of AML, B-ALL, and MM we correlated the effect of MEN1 expression to the overall survival of patients F. The anhigh in particular, high MEN1 expression is significantly adversely correlated with the efficacy of the Total Therapy 6 (TT6) treatment protocol consisting of induction therapy with Melphalan/Bortezomib/Thalidomide/Dexamethasone/Cisplatin/Doxorubicin/Cyclophosphamide/Etoposide (M-VTD-PACE) followed by a high-dose M-VTD-PACE-based tandem transplant [In MM (MMRF-COMPASS study), the overall survival of patients with higher MEN1 expression was significantly worse over the course of the study. In patients with MEN1ansplant , and in ansplant  , SUP-B15 (BCR-ABL1), and the MLLr cell lines RS4;11 (MLL-AF4)   and KOPNMLL-AF4) . Both shMLL-AF4) A and comMLL-AF4) B.The decrease in fitness in cells transduced with the MEN1-targeting shRNAs was associated with the induction of apoptosis, which was stronger in MLLr-negative cell lines C. Notabl50 > 20 µM), whereas the IC50 values of the MLLr cell lines RS4;11 and KOPN8 were 35 nM and 15 nM, respectively   D. To add> 10 µM) E.Thus, MEN1 depletion but not the inhibition of MEN1–KMT2A interaction induces apoptosis in MM cell lines. Importantly, MEN1 depletion might downregulate KMT2A protein expression in some subtypes of MM.The observed cytotoxic effect of MEN1 KD in the cell lines that do not harbor MLLr or other known mutations that endow sensitivity to the MEN1 binders was too surprising to be accepted without additional proof. Neither the RNA interference, which we used for MEN1 KD nor CRISR/Cas9-dependent DNA editing, which was used in gene-wide loss-of-function screening, which we used in our analyses, are free of off-target effects, potentially confounding the interpretation of the results ,25. In aGiven that CRISPR editing is prone to off-target effects , we chosTo analyze the effect of the MEN1 KO on cell fitness, we monitored the dynamics of knockout alleles in the whole cell population. To this end, we calculated the knockout score (percentage of edits that result in a frameshift or an indel more than 21 bp in length) at different time points after transfection. We preferred this dynamic approach to the end-point measurement (as in the case of CRISPR/Cas9 genome-wide loss-of-function screenings) and to the selection of single KO clones due to the inherent tendency of CRISPR/Cas9 DNA editing to induce TP53-dependent DNA damage response followed by growth arrest and apoptosis . Given tTransfection of the RNP complex targeting the first coding exon of MEN1 A efficieWe concluded that MEN1 depletion confers a selective disadvantage in B-ALL and MM cell lines which do not harbor MLLr, or other mutations conferring dependency on MEN1.In this proof-of-principle study, we addressed the feasibility of MEN1 depletion vs. specific targeting of the MEN1–KMT2A interaction for the treatment of lymphoid malignancies, including those that are not driven by MLLr and other known mutations endowing sensitivity to the inhibitors. We found that in contrast to the inhibition of the MEN1–KMT2A interaction, the depletion of the MEN1 protein is toxic for B-ALL cell lines independently of the driver mutations. Moreover, we corroborated the data of genome-wide loss-of-function screenings on the dependency of MM cell lines on MEN1 and showed that in some cases the depletion of MEN1 is associated with a decrease in KMT2A expression on the protein level.We have proven that cell lines that harbor neither MLLr nor other mutations conferring sensitivity to the inhibitors are dependent on MEN1 expression. This is in line with the reported high selectivity of VTP50469 , towardsMEN1 is involved in the assembly of multiple transcription-regulating complexes, and can both activate or repress transcription recruiting either epigenetic activators or repressors . MEN1 biOur data on the dependency of MM on MEN1 are of specific interest because MLLr and related mutations are not common in MM . Our datImportantly, selective inhibition of the MEN1–KMT2A or MEN1–KMT2A–FP interaction is considered a guarantee of the low toxicity of the inhibitors . On the Most recently, preliminary data on the antitumor effect of the new irreversible covalent MEN1 binder BMF-219  have beeOur study also points to protein degradation as a prospective approach to developing new drugs targeting MEN1. The stability of proteins can be selectively targeted by proteolysis targeting chimeras (PROTACs), a new group of drugs, which allow selective degradation of proteins by targeting them to the ubiquitin-proteasomal system. PROTACs consist of two ligands joined by a linker. One ligand, the “warhead”, binds a protein of interest, while the other recruits an E3 ubiquitin ligase which transfers ubiquitin to the protein of interest to facilitate its proteolytic degradation . Given tConclusively, our study provides the rationale for targeting MEN1 for degradation, for example by PROTACs. According to our data, this approach would be more efficient and have a broader antitumor spectrum than specific inhibitors of MEN1–KMT2A interaction. It is conceivable that changing the paradigm of the design of MEN1 binders towards compounds that remodel physiological MEN1 PPIs or facilitate the building of neo-PPIs will reveal a new, hitherto unknown potential of MEN1 targeting.Additional and detailed information on all experimental procedures and reagents is provided in the 2.B-ALL cell lines NALM6, RS4;11, SUP-B15, were cultured in complete RPMI1640 medium containing 20% of FCS , whereas KOPN-8 in complete medium containing 10% FCS. MM cell lines U266; OPM2, and MM1.s were cultured in RPMI medium containing 10%, for KMS-12-BM 20%, for L363 15%. MM LP1 cells were cultured in IMDM medium  containing 20% FCS. The HEK293T cells were cultured in complete DMEM medium (Gibco) con-taining 10% FCS. In addition to FCS complete medium was always complemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 50 µM monothioglycerol for the MM cell lines. All cells were cultured at 37 °C and 5% COhttps://www.broadinstitute.org/rnai-consortium/rnai-consortium-shrna-library, accessed on 2 March 2022). Corresponding oligonucleotides were assembled and cloned into pRSI12-U6-sh-UbiC-TagRFP-2A-Puro vector . ShRNA target sequence as well as amplification primers are listed in the The shRNA target sequences for MEN1 were selected from Broad Institute RNAi consortium shRNA library . The crRNAs, tracrRNA, and Alt-R® S.p. Cas9 Nuclease V3 were obtained from IDT . To assemble RNP complexes 200 µM of each tandem crRNA and 200 µM tracrRNA were mixed at equimolar ratio, heated at 95 °C 2 min and cooled down at room temperature. Once the mix has cooled, 450 pmol of each of the two different crRNAs:tracrRNA complexes were added to 150 pmol in total of Cas9 per nucleofection. The RNP mix was incubated in the dark at room temperature for 15min. In the meantime, 5 × 106 cells werte washed once in PBS and centrifuged at 1200 rpm for 5 min. The cell pellet was resuspended in 100 µL of corresponding nucleofection buffer and transfected with help of 4D-Nucleofector® X- . Typically, 5 × 106 cells were nucleofected with RNP complexes using 100 µL cuvettes. Nucleofected cells were incubated in complete medium at normal conditions. Medium was changed every three days to maintain cell density of 0.5–1.0 × 106 per ml. Starting from the day 4 the cells were harvested with 7 day intervals. gDNA was extracted from up to 10 × 106 cells with help of DNeasy Blood & Tissue kit . To amplify edited regions, we used PCR primers targeting flanking sequences. The primers were synthesized by Biomeres.net GmbH . The gDNA was amplified with help of GoTaq DNA Polymerase  in the presence of 5% DMSO and the amplified fragments we visualized on 1% agarose gel and purified with help of QIAquick gel extraction kit (Qiagen). The DNA fragments were sequenced by Eurofins . To assess the spectrum of the InDels, the Sanger chromatograms were analyzed with help of Inference of CRISPR Edits (ICE) CRISPR Analysis tool  Synthego Corporation . This method confer analysis of the indels, generated by CRISPR-editing at the resolution level of next generation sequencing. To assess dynamics of potentially inactivating indels we used ICE parameter “Knockout Score”, which represents the proportion of cells that have either a frameshift or indel exceeding 21 bp. To control potentially pro-apoptotic effect of CRISPR-induced double strand break or to exclude selection of TP53-deficient clones, which is an intrinsic setback of CRISPR-dependent DNA editing, as control we edited 3rd intron of TNNI3.Tandem CRISPR/Cas9-dependent gene inactivation was performed similarly as originally described  with somImmunoblotting was done as we described previously . Primers50 IC50 by MTT test was done over the course of five days as we described previously [4 cells per well in complete medium. Solvent control wells were incubated with dimethyl sulfoxide (DMSO). Positive control wells were treated with 5 µg/mL puromycin . Cells were incubated at standard conditions for 5–6 days, followed by addition of 25 µL of the 5 mg/mL MTT solution (Sigma-Aldrich) for 2 h at normal culture conditions. Then, 100 µL lysis buffer  was added and after an overnight incubation at 37 °C, the optical densities (OD) were measured at 570 nm wavelength using the SpectraMax 250 microplate reader  with help of the SoftMax Pro 3.0 software . Percentage of growth inhibition at a given drug concentration was calculated as (1 − ODdrug − ODpuromycin)/ODDMSO × 100. The half maximal inhibitory concentration was calculated using GraphPad Prism software (RRID: SCR_002798). For competitive growth assay percentages of RFP+ cell populations were analyzed using the BD FACSDiva Software (RRID:SCR_001456) or Flow Jo Software . The percentage of RFP+ cells was measured every 3 days. First measurement was performed 4–5 days post transduction and the percentage of transduced cells was set as 100% and used for normalization of results of following measurements.Apoptosis, cell viability, and determination of ICeviously . To assehttps://depmap.org, accessed on 28 February 2023) database which comprises data on genome-wide loss-of-function CRISPR/Cas9 screenings. For the analysis we included all AML, B-ALL, and MM cell lines available in the database at that time of analysis. DepMap calculates “gene effect scores” to measure the effect of knocking out of the gene on the viability of a given cell line. These values are calculated using the Chronos algorithms [https://depmap.org, 6 August 2022). The calculation of IC50, and statistical analyses of the data using Mann-Whitney-Test and regression analysis were done with GraphPad PRISM .CRISPR dependency data for MEN1 were mined from DepMAP (DepMap 22Q4, Chronos, gorithms . The dat


Elraglusib (formerly 9-ING-41) is an ATP-competitive inhibitor of glycogen synthase kinase-3β (GSK3β) undergoing clinical trials for the treatment of various cancers including non-Hodgkin lymphoma (NHL). The drug reduces proliferation of several NHL cell lines and has efficacy in xenograft models of the disease. To confirm the importance of its action on GSK3β, we treated 3 lymphoma cell lines with selective, structurally distinct GSK3 inhibitors: CT99021, SB216763, LY2090314, tideglusib, and elraglusib. Stabilization of β-catenin and reduced phosphorylation of CRMP2, two validated targets of GSK3, were used as functional read-outs for GSK3 inhibition. CT99021, SB216763, and LY2090314 failed to reduce proliferation or viability in any cell line at concentrations that stabilized β-catenin and reduced CRMP2 phosphorylation. There was partial reduction of CRMP2 phosphorylation but no significant effect on β-catenin at cytotoxic doses of elraglusib. There was no indication of GSK3 inhibition at doses of tideglusib that affected cell viability and apoptosis. Cell-free kinase screening confirmed several other targets of elraglusib, distinct from the GSK3 inhibitors with no anti-lymphoma actions, including PIM kinases and MST2. These data question GSK3 as the target of elraglusib in lymphoma, and hence the utility of GSK3 expression as a ‘stand-alone’, therapeutic biomarker in NHL.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-023-01147-8. Despite improvements in outcomes, high-grade B- and T-cell non-Hodgkin lymphoma (NHL) represent a significant area of unmet therapeutic need, particularly in the context of relapsed or refractory disease , 2.Glycogen synthase kinase 3 (GSK3) is a ubiquitously expressed, serine/threonine kinase involved in diverse cellular functions . Two parA recently developed GSK3 inhibitor, elraglusib (formerly 9-ING-41), is particularly noteworthy for its anti-lymphoma effects in both B- and T-cell NHL lines and immunocompromised mouse patient-derived lymphoma xenograft models , 12.Elraglusib has now progressed to early-phase clinical trials for several malignant conditions, including lymphoma, with clinical efficacy reported in case studies of refractory adult T-cell leukemia/lymphoma , 14. TheDoxorubicin, SB216763, elraglusib (9-ING-41) and tideglusib were purchased from Cambridge Bioscience, LY2090314 from Stratech, CT99021 was synthesized as previously described . All comHS-Sultan cells were gifted from Dr Banerjee, University of Dundee. Karpas-299 cells were purchased from Public Health England and HH cells from American Type Culture Collection. Cells were grown in RPMI-1640 media containing glycine, 10% fetal bovine serum supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin .To measure changes in protein expression, cells in the exponential phase of growth were cultured in serum-free media overnight, prior to 4 h of drug treatment. For PARP (Poly [ADP-ribose] polymerase) cleavage assays, cells were cultured in serum-containing media for 24 h prior to protein extraction .Expression of β-catenin and phosphorylation of collapsin response-mediator protein 2 (CRMP2) were used as functional read-outs of GSK3 inhibition and PARP cleavage for apoptosis, all measured by immunoblotting. Rabbit anti-PARP (9542), rabbit anti-CRMP2 (9393) and rabbit anti-β-catenin (9562) were from Cell Signaling. Mouse anti-actin (A3853) was from Sigma-Aldrich. Sheep anti-phospho-CRMP2 (Thr509/514) was made in-house as described previously .p =  < 0.05 for statistical significance.Anti-rabbit (Licor Goat anti-Rabbit (926–32211)) and anti-mouse (Thermofisher Invitrogen Goat anti-Mouse (A21057)) secondary antibodies were used at 1:10,000. Rabbit anti-sheep HRP (horseradish peroxidase) secondary (ThermoFisher 31480) was used at 1:2500. All antibodies were diluted in 1% BSA in TBS-T. Cell lysates were prepared for SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) as previously described , and proThis was assessed using CellTiter 96® Aqueous One Solution Cell Proliferation Assay Kit (Promega) according to manufacturer’s instructions. In brief, 8000 cells/well were seeded into 96-well plates. Compounds were incubated with cells for 72 h prior to the addition of MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reagent for 2 h followed by absorbance assessment at 450 nM by Modulus Microplate reader. Analysis was performed using Graphpad Prism 9.33P cell-free screens were performed by the MRC Protein Phosphorylation Unit, University of Dundee as previously described [escribed .Elraglusib 1-5 µM) reduced cell viability and induced apoptosis in both B- and T-cell NHL cell lines in keeping with its reported anti-lymphoma properties  produced a weak reduction in cell viability and only induced apoptosis in one of three cell lines, while none of the other GSK3 inhibitors reduced cell viability or induced apoptosis at concentrations previously reported to result in GSK3 inhibition  at concentrations that generate changes in cell viability.TP53-mutant Burkitt lymphoma cell lines to anthracycline chemotherapy [Wu et al. were unable to generate GSK3β knockouts in four of five lymphoma cell lines, an intrinsic dependence upon GSK3β for lymphoma survival was suggested [These are important observations as GSK3 is increasingly recognized as a potential therapeutic target in hematological malignancy. Structurally distinct GSK3 inhibitors can induce apoptosis in patient-derived CLL cells, reduce proliferation in AML cell lines and re-sensitize otherapy –11. Sincuggested . These duggested . Howeveruggested –24. Simiuggested .In conclusion, we re-affirm the anti-lymphoma effects of elraglusib but argue that this action cannot be attributed to GSK3 inhibition. Hence, we caution against the assumption that selective GSK3 inhibitors will have clinical benefit in lymphoma and against GSK3 activity as a mechanistic biomarker.Additional file 1.


This review paper is focused on the design of anthracene and furan-containing Schiff bases and their advanced properties as ligands in complex transition metal ions The paper also provides a brief overview on a variety of biological applications, namely, potent candidates with antibacterial and antifungal activity, antioxidant and chemosensing properties. These advantageous properties are enhanced upon metal complexing. The subject of the review has been extended with a brief discussion on reactivity of Schiff bases with hydrogen phosphonates and the preparation of low and high molecular phosphonates, as well as their application as pharmacological agents. This work will be of interest for scientists seeking new challenges in discovering advanced pharmacological active molecules gaining inspiration from the versatile families of imines and aminophosphonates. Schiff bases present an important class of compounds that contain the azomethine (–C=N–) group in their structure. They are formed by the reaction between a primary amine and an aldehyde or a ketone under specific conditions. Schiff bases belong to the versatile family of imines. The general structure of Schiff bases and the synthetic path to their formation are presented in Though other ways of obtaining Schiff bases have been invented, the reactions between amines and either aldehydes or ketones present the most exploited method. While the aldehydes react with primary amines upon mixing to form Schiff bases, the reaction with ketones depends on the type of catalyst, pH range, the type of solvent and the reaction temperature .One of the most important properties of Schiff bases is their selectivity toward metal ions and ability to form complexes, including highly stable 4-, 5- and 6-ring complexes. Just like the Schiff bases, their metal complexes also possess anticancer, antibacterial, antifungal and antiviral properties ,8,9,10. For the last decades, a lot of research groups have been investigating Schiff bases and reporting on the huge scope of bioactivities these compounds possess–antiviral ,12, antiThe variety of structures and properties broadens the application of Schiff bases covering chemical catalysis and chemical analysis, diagnosis and therapy, new technologies such as television and computer screens, digital clock displays, etc. ,23,24,25The combination of a carbon nitrogen double bond with heteroatom rings leads to the enhancement of most of the above-mentioned abilities of the Schiff bases . Such coIncreasing knowledge of cancer chemotherapy, it is needful to design new compounds to overcome the resistance of available anticancer drugs. α-Aminophosphonates are an important class of compounds that have received wide attention due to various pharmaceutical applications such as anticancer, antiviral, antibacterial drugs, antibiotics, etc. ,29,30. AThree outstanding scientists Kabachnik, Medved and Fields discovered in 1952 independently the reaction known nowadays as the Kabachnik–Fields reaction  followinAnother known reaction for C–P bond formation is the Pudovik reaction, which is often named as the phospha-Michael addition . It is oOn the basis of our scientific experience in organic and polymer synthesis in collaboration with colleagues from biological and pharmaceutical sciences, the idea arose to combine in a joint review paper results of research that cover the synthesis of biologically active Schiff bases and their transformation into low- and high-molecular aminophosphonates with determination and analysis of their biological activity. To the best of our knowledge, such summary material has not been published, and we believe that it will be useful both for those studying and working in the field of organic, polymer and medicinal chemistry, as well as in the design of drugs, macromolecular biodegradable compounds with intrinsic bioactivity, development of therapeutic and diagnostic systems.Bacillus thuringiensis, Streptococcus pneumoniae, Escherichia coli and Pseudomonas putida compared to the free ligand  via a condensation reaction between furan-2-carboxaldehyde and propane-1,3-diamine. The novel bis-Schiff base was used as ligand in the complex formation with a number of transition metal ions (Co(II), Ni(II), Cu(II), Mn(II), Cd(II), Zn(II) and Fe(III)) which demonstrated higher antimicrobial effect than the free bis-Schiff base as shown in According to the literature, not much scientific works have been reported on the preparation of complexes of transition metals with Schiff base derivatives of 2-aminobenzamide providing oxygen and nitrogen donors. Therefore, Tyagi et al. presented in their research the synthesis of a Schiff base derived from the reaction of 2-aminobenzamide with furan-2-carboxaldehyde and their complexes with Ni(II), Cu(II) and Co(II) metal complexes . They weA Schiff base (Z)-4-((furan-2-ylmethylene)amino)benzenesulfonamide was synthesized via a reaction between furfural and sulfanilamide. The new compound exhibited a moderate antimicrobial activity compared to a standard drug. A molecular simulation was performed on the compound attached to a human mitochondrial (2WYA) protein and an excellent binding affinity score was observed .3+ in ecological and biological systems. Since aluminum is one of the most abundant elements on the planet, it is extensively used by many industries, thus polluting the environment and harming the human body. Fluorescent detection technique has been often used for the detection of Al3+, but it has also some limitations, such as the complicated synthetic procedures for the preparation of the samples, long response time and poor selectivity. To overcome these limitations Peng et al. prepared a Schiff base derived from furan- 2-carbohydrazide and 2-hydroxy-4-methoxybenzaldehyde . The product exhibits excellent selectivity and sensitivity to Al3+ with turn-on fluorescence in a water environment. Furthermore, the detection limit of the ions was very low and a practical application was found for PEGFB in preparing test strips coated with PEGFB to be used in aqueous environment toluidine;N,N-dimethyl-[N’-methyl(diethoxyphosphonyl)--(2-furyl)]-1,3-diaminopropane .The starting furan-containing Schiff bases and their a-aminophosphonates were evaluated for antiproliferative activity against four human leukemic cell lines, together with the multi-drug-resistant model HL-60/Dox. All compounds exerted concentration-dependent cell growth-inhibition effects after 72 h exposure .b cell line and moderate activity against LAMA-84 and multi-drug-resistant phenotype of the acute promyelocyte leukemia (HL-60/DOX) cell line -1,3-diaminopropane as furan-containing aminophosphonate was investigated for in vitro cytotoxic activity on six human epithelial cancer cell lines. Testing for Safety was conducted in vivo on ICR mice and in vitro (3T3 NRU test) for antiproliferative activity and genotoxicity. The aminophosphonate demonstrated high in vitro cytotoxic activity against the 647-V cell line (human bladder carcinoma) and the HepG2 cell line (human hepatocellular carcinoma). This furan-containing aminophosphonate possessed a moderate genotoxic and antiproliferative activity in vivo and presented a complete absence of toxicity to Balb/c 3T3 (clone 31) mouse embryo cells -p-toluidine, [N-methyl (diethoxyphosphonyl)-1-(9-anthryl)]furfurylamine and [N-methyl (dimethoxyphosphonyl)-1-(9-anthryl)]-p-toluidine.Looking for efficient synthetic procedures to obtain these valuable biologically active compounds, we applied microwave synthesis as an alternative to the classical method . For theWe found out that microwave synthesis has a lot of benefits, as these synthetic procedures result in high yield under mild conditions and in short reaction times. We separated enantiomers of two racemic α-aminophosphonates with a chiral center and analytical and semi preparative separations were optimized. The high enantiomeric purity of the isolated enantiomers was appropriate for pharmaceutical investigations. The racemic substances and their enantiomers were investigated for cytotoxicity and genotoxicity in vivo. All investigated substances demonstrated a weak genotoxic effect. Almost half retains the proliferative capacity of the bone marrow cell population compared to the untreated control. The results allow us to assume that the hematopoietic function of the bone marrow will not be discontinued after exposure to the investigated substances .50 values shown in These Schiff bases and their derived aminophosphonates were studied for cytotoxicity against a panel of seven cancer cell lines representative of some important species of human tumors. All substances showed dose-dependent cytotoxicity after 24 h of treatment which enabled the creation of the dose-effect curves and the calculation in accordance with the equieffective ICA Schiff base containing furan and anthracene rings and aminophosphonate derived from it showed twofold higher activity towards HBL 100 cells than the control drug Doxorubicin. The antiproliferative potential of these substances was comparable to that of the positive control substance used in the experiments when tested on cell lines 647-V (bladder carcinoma) and MDA-MB-231 (highly metastatic carcinoma of the breast). All tested substances demonstrated less activity than the control drug Doxorubicin after experiments with cell cultures from HeLa, HepG2 and MCF-7 cancer cell lines .The presence of both furan and anthracene rings is essential for the optimal activity of these compounds in the chemotherapy of malignant breast and colon disease .p < 0.001) cytotoxicity in a wide concentration range (1–0.07 mg/mL) to mouse embryo fibroblastic cells compared to not treated control cell cultures. However, compared to the cytotoxic effect of the positive control substance sodium dodecyl sulphate on Balb/c 3T3 cells, the cytotoxicity of the tested compounds was comparable [The results from the validated Balb/c 3T3 (clone 31) Neutral Red Uptake Assay (3T3 NRU test) revealed dose-dependent cytotoxic activity of investigated substances. The Schiff base containing anthracene and furan rings and its corresponding aminophosphonate showed statistically significant -1-(9-anthryl)]-p-toluidine was found in the nuclei and nuclear membranes of HBL-100 cells  77]..77].N-methyl(diethoxyphosphonyl) -1-(9-anthryl)]benzidine (3) and 4.4′-bis[N-methyl(diethoxyphosphonyl)-1-(9-anthryl)]diaminodiphenylmethane (4) (Our research continued with investigation of new bis-aminophosphonates: bis. Th. Th90]. The poly(aminophosphonate)s 9 and 10 were obtained in similar manner from a Schiff base N,N-dimethyl-N’-furfurylidene-1,3-diaminopropane and poly(oxyethylene H-phosphonate)s derived from PEG200 and PEG600 .50 values, whereas polymer 9 was less active, and 5 exhibited only marginal toxicity.The poly(oxyethylene aminophosphonate)s 5–10 have coordination centers in their repeating units and are candidates as new biodegradable polymer carriers for the physical conjugation of bioactive compounds. The polymers were evaluated for cytotoxicity in four malignant cell lines following 72 h treatment at varying concentrations . Polymer8 were identified as structural prerequisites affording superior activity. The analogues obtained from the Schiff bases N-(4-dimethylaminobenzylidene)-p-toluidine and N,N-dimethyl-N’-furfurylidene-1,3-diaminopropane displayed lower activity than 8. Despite the Schiff base residue, however, in all subseries of poly(aminophosphonate)s, the decrease in the length of the PEG segments (from 13 oxyethylene units in PEG600 to 4 units in PEG200) evoke a noticeable reduction in relative potency, most prominent in the case of 5 where almost total loss of activity was observed. The cytotoxicity of polymers 6, 7, 8 and 10 was found to be comparable to the reference drug cisplatin, i.e., findings that gave us reason to consider them as a novel class of aminophosphonate-delivered cytotoxic substances s.In the copolymers, the content of the hydrophilic H-phosphonate units is greater than the aminophosphonate fragments, which makes them soluble in water. The fluorescence spectra of all polymers revealed the emission maxima in the blue—blue-green spectral range.The copolymers were evaluated for in vitro cytotoxicity on a panel of seven human epithelial cancer cell lines.These new compounds appeared to belong to the low-toxicity group of DNA intercalators. Investigated polymers appear promising for the development of active antineoplastic drugs for chemotherapy of malignant breast and liver disease.The results of the fluorescent microscopy evaluation well paralleled the results of in vitro cytotoxicity and revealed apoptotic and necrotic alterations in breast tumor cells and hepatocellular carcinoma cells after prolonged exposure, which gives insights on the potential mechanism of cancer cell eradication. The clastogenic effects of the elaborated polymers have been reduced over time. Although, the tested polymers showed only slight suppression on bone marrow cell division, as compared to the referent drug (Mitomycin C), they significantly inhibit the mitotic processes compared to the untreated cells .In summary, we have studied the methods of obtaining the anthracene-derived Schiff base S-1 and anthracene and furan-containing Schiff base S-2, α-aminophosphonates A-3–A-6, bis-aminophosphonate B-6 , and polyphosphoesters P-8–P-11, bearing aminophosphonate fragments .Anthracene-delivered Schiff base S-1 and the corresponding α-aminophosphonates A-3 and A-4 and the polyphosphoesters P-8–P-11, and anthracene and furan-bearing Schiff base S-2 and its derivatives, the substances A-5 and A-6, two enantiomers A-5a and A-5b were investigated for antiproliferative activity against human leukemic cell lines, exploiting cisplatin as a control anticancer drug.Among the investigated substances A-3, A-4 and A-5, aminophosphonate A-5 with anthracene and furan rings in the structure, displayed the highest inhibitory activity against all studied human epithelial cancer cell lines. This result motivated us to introduce aminophosphonate building blocks with anthracene and furan residues into the polyphosphoester backbone to yield novel polyaminophosphonates P-12 and P-13. They possess hydrolytically unstable phosphoester linkages in their backbones. The polymers P-8 and P-10 are built only of aminophosphonate repeating fragments, while the copolymers P-9 and P-11–P-13 are composed of polyoxyethyleneaminophosphonate blocks and hydrophilic polyoxyethylene H-phosphonate segments. The antiproliferative potential of the new macromolecules was tested against a series of human cancer cell lines: K-562, SKW-3, REH and HL-60 .The results indicate that aminophosphonate A-4 and polymers P-8 and P-9 could be developed as promising new candidates for cytotoxic agents .Schiff Bases and their derivatives as aminophosphonates and poly(aminophosphonate)s are capable of inhibiting tumor cell growth and inducing cell death in different test systems, necessitating further exploration for the delineation of their antineoplastic potential. Furthermore, the presence of anthracene and furan-containing moieties impart additional valuable properties such as fluorescent activity and enhanced penetration via biological membranes beneficial in the design of novel pharmaceutical agents. Polyphosphoesters are appropriate macromolecules for developing drug delivery systems as they are biodegradable in a physiological medium and yield biocompatible and non-toxic degradation products. The availability of pharmacophore fragments and a biodegradable and non-toxic polymer backbone leads to obtaining macromolecular drug carriers with their own pharmacological activity and opens an investigational avenue for the future exploration thereof.


BackgroundCancer is the second most common cause of death. Oral squamous cell carcinoma (OSCC) represents the most frequent of all oral neoplasms. Many treatment modalities such as chemotherapy, radiotherapy, surgery, and immunotherapy are emerging but still, the patients' quality of life is questionable. Despite the advances in therapeutic approaches, the percentages of morbidity and mortality of OSCC have not improved significantly during the last 30 years. Treatment using natural products can act as a potent anti-cancer agent with reduced adverse effects. Cinnamic acid derivatives exhibit anti-cancer potential through histone deacetylase inhibitor (HDAC) enzyme inhibition.MethodologyIn an experimental study design, cinnamoyl hydroxamate derivatives were prepared. The structure was confirmed using ultraviolet-visible spectroscopy (UV-Vis), nuclear magnetic resonance (NMR), infrared spectroscopy, and mass spectrophotometry. An in-vitro antioxidant assay using nitric oxide scavenging and reducing power assay was done and an in-vitro cytotoxic  (MTT) assay and viability assay were carried out using tryphan blue dye.ResultsStatistical analysis was performed using SPSS . Cinnamoyl hydroxamate derivatives were obtained and named as compounds 3a (E)-N-Hydroxy-3-(4-(N-(phenyl bromo) sulfamoyl) phenyl) acrylamide-) and 3b ((E)-N-Hydroxy-3-(4-(N-(phenyl nitro) sulfamoyl) phenyl) acrylamide). In the nitric oxide scavenging assay, compound 3a showed good antioxidant activity than 3b. Reducing power assay was higher in 3a compared to 3b. Cell viability using tryphan blue exhibited a concentration decrease in % cell viability with an increase in the concentration of human oral cavity squamous cell carcinoma cell line (OECM 1), a unique head and neck squamous carcinoma cell line (UM SCC 6) & human oral squamous cell carcinoma forming metastatic foci (HSC 3) cell lines.ConclusionThe results of the present study revealed that the study compounds play a vital role in the up-regulation of apoptotic pathways and regulation of terminal differentiation pathways. The compounds showed good anti-oxidant and anti-cancer activities in lesser concentrations, hence they can be used as a therapeutic agent for oral squamous cell carcinoma. The major global cause of adult mortality is cancer. The term "oral cancer" refers to a diverse range of cancers that can develop in the mouth in various locations. More than 4.5 million people worldwide pass away from cancer each year, with an estimated nine million new cases being diagnosed each year. Oral cancer is one of the top three types of cancer in India, accounting for the second and third most prevalent types of cancer in men and women, respectively .Uncertainty, exposure to harsh climatic conditions, and behavioral risk factors are signs of a broad range of incidences over the world. In addition to being a significant risk factor for oral cancer, periodontal diseases are more common in the Indian population, where the practice of chewing paan is a major contributing factor. Inflammation, which is brought on by bacterial and viral infections as well as inflammation, is involved in the development of tumors .Oral squamous cell carcinogenesis is a multi-step process in which several genetic occurrences modify the oncogenes' and tumor suppressor genes' typical roles, disrupting the pathways normally used for regulatory control. This may lead to a rise in the production of transcription factors, growth factors, or the number of cell surface receptors, as well as improved intracellular messenger signaling. This causes a cell phenotype capable of enhanced cell proliferation, with a lack of cell cohesion, and the ability to infiltrate local tissue and spread to distant areas when combined with the loss of tumor suppressor activity .Conventional diagnostic techniques like clinical and histopathological examination, vital staining, biopsy, and spectroscopic analysis are been used to detect oral cancer. But diagnosing cancer at an early stage will help in better survival and quality of life of the patients.Based on the staging of the cancer, the treatment modalities vary. Chemotherapy, radiotherapy, surgery, targeted therapy, and immune therapy are the various treatment for treating oral cancer. Each therapy has its pros and cons. Oral cancer patients undergo various physical and psychological adverse effects which can be short-term or long-term. Cancer survivors face many ill effects like recurrence or secondary tumors along with cardiovascular, renal, and lung complications. The OSCC patients treated with surgery usually have difficulties in swallowing and speech, and also treatment can lead to neuralgia, altered or complete loss of taste sensation. Surgery can also cause cosmetic disfigurement which may require further reconstruction and rehabilitation .Various Indian herbs have been used as anti-cancer agents are Curcuma longa, Zingiber officinale, Cinnamomum verum, Crocus sativus, Ocimum sanctum, Azadirachta indica, and so on. A vital substance present in plants like Cinnamomum cassia (Chinese cinnamon) and Panax ginseng is cinnamic acid, a naturally occurring aromatic carboxylic acid. Cinnamon bark is used to make cinnamic acid. Its structure, which consists of a benzene ring, an alkene double bond, and a functional group of acrylic acid, allows for the modification of the aforementioned functionalities with a wide range of substances to produce bioactive molecules with increased efficacy. It has been discovered that the type of substituents added to cinnamic acid has a significant impact on how biologically effective are synthesized cinnamic acid derivatives .Cinnamic acid possesses anti-cancer, anti-diabetic, anti-inflammatory, anti-bacterial, and neuroprotective effects. By giving the electrons that interact with radicals to produce stable products, cinnamic acid stops the radical chain reactions. Detergents, flavorings, cosmetics, and toiletries all use it as a fragrant element. Enzymatic deamination of phenylalanine can produce cinnamic acid. Derivatives of cinnamic acid have anti-cancer properties and are effective against a variety of malignancies, including breast, colon, and lung cancers. It has been noted that cinnamic compounds have anti-proliferative effects on tumors. Apoptosis is one method used by cinnamic acid derivatives, such as cinnamaldehyde, to kill malignant cells .Through the suppression of the histone deacetylase inhibitor (HDAC enzyme), derivatives of cinnamonyl sulfonamide hydroxamate show selective anti-cancer action in human cancer cells. HDAC inhibitors target microtubules and impair the control of mitotic progression, induce apoptosis, and thus are potent anti-cancer agents. Thus the present study will evaluate the anti-cancer activity of cinnamoyl sulfonamide hydroxamate derivatives against squamous cell carcinoma.The present study is an experimental research, carried out in Santosh Dental College. The material preparation and analysis are carried out in Caritas College of Pharmacy, Kottayam, and the cell culture techniques are carried out in Biogenics, Trivandrum. The study was carried out in various phases. The ethical clearance was obtained with reference 856/PO/Re/S/04/CPSEA.Design of cinnamoyl hydroxamate derivatives (E)-3-(4-(Chlorosulfamoyl) Phenyl) Acrylic Acid SynthesisChlorosulfonic acid (0.69g at 5.4 mM) and cinnamic acid  were mixed at 35 °C for four hours. To keep an eye on the reaction's development, pre-coated thin-layer chromatography (TLC) plates were employed. A beaker containing ice cubes was then filled with the viscous reaction mixture. The resulting yellow precipitate (anhydrous CaCl2) was filtered and it was washed thrice with 20 ml of distilled water and dried in a vacuum to reduce free anhydrous molecules.(E)-3-(4-(N-(Phenyl Bromo) or (Phenyl Nitro) Sulfamoyl) Phenyl) Acrylic Acid SynthesisBromo aniline  or nitro aniline  were added to a quantity of (E)-3-(4-(chlorosulfamoyl) phenyl) acrylic acid  in 50 ml distilled water, and pH 8 was maintained using aqueous NaHCO3. At 35° C, the reaction mixture was agitated for four hours. The liquid was then adjusted to have a pH of 2 by adding hydrogen chloride (HCl) drop by drop. Product three was recovered as a white precipitate after being washed repeatedly with water, dried, and then ethyl acetate is used to recrystallize.(E)-N-Hydroxy-3-(4-(N-(Phenyl Bromo) or (Phenyl Nitro) Sulfamoyl) Phenyl) Acrylamide SynthesisUsing a calcium chloride guard tube, compound (E)-N-Hydroxy-3-(4-(N-(phenyl bromo)  was added to 30 ml of dichloromethane (Cl2CH2), along with ethyl chloroformate  and N-methyl-morpholine . The reaction mixture was then agitated at 35 °C for five hours. On a pre-coated TLC plate, the full translation of acrylic acid to acid chloride was seen. Additionally, the addition of freshly made neutral hydroxylamine solution  in 30 ml of tetra-hydro-furan (THF) completed the translation of acid chloride to the corresponding hydroxamate derivative. To create the finished product, column chromatography was used to purify the recovered residue, and two compounds were retrieved  was used for further analysis.Characterization of synthesized cinnamyl sulfonamide hydroxamate derivativesStructure ConfirmationUltra violet visible spectroscopy (UV-Vis) was used to determine the purity of the compound and their absorbance spectrum as they absorb light at specific wavelengths with unique absorbance values. The extracted sample was analyzed using Shimadzu UV-1800 UV spectroscopy. Infrared spectroscopy (IR) was used to identify the molecular structure, identification of chemicals, their chemical bonds, and also their qualitative and quantitative determination of chemicals present. Mass spectroscopy (MS) was done to identify the unknown compounds by molecular weight determination, quantify the known compounds, and also to determine the chemical properties of the molecules present. It was done using Thermo-Fischer scientific mass spectroscopy. For nuclear magnetic resonance (NMR), a thermo-scientific pico spin spectrometer was used to identify the molecules, study the molecular interactions, and also to probe the molecular dynamics (1H and 13C).Derivatives of synthesized cinnamyl sulfonamide hydroxamate(E)-N-Hydroxy-3-(4-(N-(phenyl bromo) or (phenyl nitro) sulfamoyl) phenyl) acrylamide synthesis- compound 3a, 3b.Compound 3a(E)-N-Hydroxy-3-(4-(N-(phenyl bromo) sulfamoyl) phenyl) acrylamide- percentage yield: 2.37g, 64.56 %; molecular weight: 397g; retardation factor (Rf) value= 0.58; melting point= 184 ± 1 °C; soluble in organic solvents and insoluble in water.Compound 3b(E)-N-Hydroxy-3-(4-(N-(phenyl nitro) sulfamoyl) phenyl) acrylamide- percentage yield: 2.64g, 68.93 %; molecular weight: 363g; retardation factor (Rf) value= 0.86; melting point= 144 ± 1 °C; soluble in organic solvents and insoluble in water. (compound 3 was taken as it showed highly positive anti-cancer activity).To evaluate the in-vitro antioxidant activity of derivativesNitric Oxide Scavenging ActivityIn the nitric oxide scavenging assay, the sodium nitro prusside is kept in an aqueous solution, and will produce nitric oxide on its own, which will subsequently combine with oxygen to produce nitrite ions, which can be detected using Griess reagent. The reaction between oxygen and nitric oxide scavengers reduces the production of nitrite ions. Spectrophotometry was used to assess the nitric oxide scavenging activity. The reagents used were sodium nitroprusside, Griess reagent, and phosphate-buffered saline (PBS) reagent. Sodium nitroprusside (5 millimol L-1) was mixed with various amounts of samples 3a and 3b in phosphate-buffered saline pH 7.4 before being incubated at 25°C for 30 minutes. Instead of the test drug, distilled water was used as a control in an equivalent amount. After 30 minutes of incubation, 1.5 mL of the incubated solution was removed and diluted with 1.5 mL of Griess reagent . By measuring the absorbance of the chromophore produced after the nitrate was diazotized with sulphanilamide and then coupled with N-1 naphthyl ethylene diamine di-hydro-chloride at 546nm, the percentage scavenging activity was calculated in relation to the standard by using % inhibition = control- test/ control x 100.Reducing Power AssayThe total anti-oxidant effect is calculated using ferric reducing antioxidant assay (FRAP). This method is predicated on the notion that the antioxidant activity grows along with the absorbance of reaction mixtures. The antioxidant compound in the samples is measured at 700 nm by a UV-spectrophotometer with potassium ferricyanide, trichloroacetic acid, and ferric chloride . The reagents used are potassium ferric cyanide, tricarboxylic acid cycle (TCA), ferric chloride, and PBS reagent.Procedure: Various amounts of samples from concentration of 10mg/mL combined with 2.5ml of phosphate buffer and 2.5ml of 1% potassium ferric cyanide. After that, the mixture was heated at 50°C for 20 minutes. Instead of the test drug, distilled water was used as a control in an equivalent amount. After incubating, 2.5 ml of 10% TCA was added and kept for 10 minutes. After the upper layer (5 ml) was blended with 5 ml of distilled water and 1 ml of 0.1% ferric chloride, the absorbance was measured at 700 nm. Quercetin at 10mg/Ml was used as a reference. In-vitro viability, cytotoxicity of synthesized compoundsViability AnalysisTryphan blue dye exclusion assay: The tryphan blue dye exclusion test can be used to count the number of live cells present in a cell solution. It is predicated on the notion that live cells will have intact cell walls which block the passage of specific dyes like propidium, Tryphan blue & eosin but dead cells do not. When a dye is merely introduced to a suspension of cells without first visually determining whether the cells are absorbing or rejecting the dye. Viable cells have transparent cytoplasm, while non-viable cells have blue cytoplasm. Preparation of phosphate buffered saline pH 7.4 0.19g of sodium dihydrogen phosphate (NaH2PO4) was mixed with 2.38g 0f disodium hydrogen phosphate (Na2HPO4) and mixed with 8g of sodium chloride and made up to 1000ml with distilled water.Preparation of cell culture: Human oral cavity squamous cell carcinoma cell line (OECM 1) & a unique head and neck squamous carcinoma cell line (UM- SCC 6) & human oral squamous cell carcinoma forming metastatic foci (HSC-3) cell lines procured from the oral cavity of female Swiss albino mice were used in the study. After 14 days of development, multiplication, and maturation of tumor cells, 5-6 mL of oral mucous fluid was aspirated using an 18G needle and transferred to a centrifuge tube containing PBS and centrifuged for 15 minutes at 800 rpm or at a higher speed for few minutes to avoid increased dead cell count. The pellet was re-suspended in fresh PBS and the process was repeated three times, sufficiently diluted, and used for the study.Preparation of 0.1% tryphan blue dye: 10 mg tryphan blue dissolved in 100 mL of normal saline. The solution was stored light in an amber color bottle at 40 C. Sample preparation 10 mg of extract was dissolved in 1mL DMSO (sample A). Step I - preparation of higher concentrations of 3a and 3b: 200μg/mL: 780μL of PBS was mixed with 20μL of sample A and 100μL cell line and 100μg/mL: 790μL of PBS was mixed with 10μL of sample A and 100μL cell line. Step II - Preparation of lower concentrations of 3a and 3b: 100μL of sample A was diluted with 900μL of DMSO (sample B) and the following lower concentrations were prepared. 20μg/mL: 780μL of PBS was mixed with 20μL of sample B and 100μL cell line. 10μg/mL: 790μL of PBS was mixed with 10μL of sample B and 100μL cell line. 5μg/mL: 795μL of PBS was mixed with 5μL of sample B and 100μL cell line. Preparation of control 900μL of PBS was mixed with 100μL cell line. The cell suspension was contained in the control tube. They were mixed and incubated for three hours at 370C and 0.1ml of the assay mixture was mixed with 0.1ml of tryphan blue dye and then the percent of non-viable cells was evaluated. The cells were counted using a hemocytometer. The percentage of viability was calculated by dividing the number of viable cells by the total number of cells and multiple by 100.Analysis of cytotoxicityMTT AssayThe MTT assay was carried out using OECM 1, UM-SCC 6 & HSC 3 cell lines. The various reagents used are MTT reagent, dimethyl sulfoxide (DMSO), and Dulbecco's modified eagle medium (DMEM). The OECM 1 and UM- SCC 6 and HSC 3 cells were cultured in DMEM. Cell lines were grown in a 25 cm2 tissue culture flask with an antibiotic solution containing amphotericin, penicillin, and streptomycin as well as DMEM supplemented with 10% FBS, L-glutamine, and sodium bicarbonate. Cultured cell lines were maintained at 37°C in an incubator with humidified 5% CO2. Through direct observation of the cells using an inverted phase contrast microscope and the MTT assay method, the vitality of the cells was assessed.Materials required: OECM 1, UM- SCC 6 & HSC 3 Cell lines, MTT reagent, DMSO, DMEM medium.Procedure: Seeding of cells in a 96-well plate trypsinized two-day-old confluent monolayer of cells was sown in 96-well tissue culture plates at a density of 5 x 104 cells per well. The cells were then incubated at 37 °C on a humidified 5% CO2 incubator.Compound stock preparation: Using a cyclomixer, 1mg of each of the test substances 3a and 3b were weighed and dissolved in 1mL DMEM. To guarantee sterility, a 0.22 m Millipore syringe filter was used to filter the sample solution. After 24 hours, the growth media was withdrawn for the anticancer evaluation. Five freshly produced compounds in 5% DMEM were serially diluted two folds five times . After receiving treatment for 24 hours, the complete plate was examined under an inverted phase contrast tissue culture microscope for an anticancer assay, and microscopic observations were captured as photographs. Cellular morphological alterations like rounding or shrinkage, granulation, and vacuolization in the cytoplasm were all taken into account as indications of cytotoxicity.Statistical analysis was performed using SSPSS . A chi-square test was carried out to check for any statistical difference between the observed value and the expected value among the control and study group. One-way ANOVA analysis was carried out to compare the means and determine the association between the means of the study group. Post-hoc Tukey HSD (beta) was performed to facilitate pairwise comparisons within the analysis of variance (ANOVA) data. The level of significance [P-Value] was set at P<0.05.Cinnamyl sulfonamide hydroxamate derivativesCompound 3a(E)-N-Hydroxy-3-(4-(N-(phenyl bromo) sulfamoyl) phenyl) acrylamide- percentage yield: 2.37g, 64.56 %; molecular weight: 397g; retardation factor (Rf) value= 0.58; melting point= 184 ± 1 °C; soluble in organic solvents and insoluble in water.Compound 3b(E)-N-Hydroxy-3-(4-(N-(phenyl nitro) sulfamoyl) phenyl) acrylamide- percentage yield: 2.64g, 68.93 %; molecular weight: 363g; retardation factor (Rf) value= 0.86; melting point= 144 ± 1 °C; soluble in organic solvents and insoluble in water.Structural characterization of synthesized cinnamyl sulfonamide hydroxamate derivativesThe C=O stretching was present at 1672, the C-S group was present at 672 cm-1, and the presence of the O=S=O group was indicated by a peak at 1158 cm-1 in the FTIR spectra of the 3a. O-H and N-H groups are present, as shown by the peaks at 3260 and 2676. A peak at 3360 cm-1 and another at 3327 cm-1 in compound 3b's FTIR spectrum suggest the existence of O-H and N-H groups, respectively. Peaks for C=O at 169 cm-1 and for C-S at 697 cm-1 are also produced by the compound. The presence of the S=O group is indicated by the peak at 1179 cm-1. The synthetic compounds 3a and 3b's 1H NMR spectra reveal the existence of 10 aromatic protons in the 6-8 ppm region. Both compounds had a peak for N-H protons at 10 ppm. The synthetic compounds 3a and 3b's 13C NMR spectra reveal the existence of C=O at a peak value of 167.50 and 168.11, respectively. The ranges of 124.44-142.37 and 14.36-129.12, respectively, contain the peaks in 3a and 3b showing the presence of aromatic carbons, respectively. The molecular ion peak may be seen as the base peak at m/z 398 and 365 in the mass spectra of compounds 3a and 3b. Several spectroscopic analytical techniques were used to confirm the structure of the synthesized molecules.Nitric oxide scavenging assayAnti-oxidant activity can be analyzed by determining the nitric oxide radical inhibition concentration carried out by 3a and 3b (two synthetic derivatives) with gallic acid as standard at reductive potential concentration Table . The dosThe percentage inhibition of the standard gallic acid increased with increasing concentration of the compound assay and showed maximum inhibition of 95.7% at 200 μg/mL concentration and a minimum of 17.5% at 6.25μg/mL concentrations. Correspondingly, for both test compounds 3a and 3b, the inhibition (%) percentage scavenging increases linearly with concentration at the given cell phase. The half-maximal inhibitory concentration . This examines the cell suspension depending on the integrity of the cellular membrane exposed to compounds 3a and 3b in the present study with the percentage viability expressed as mean± SD Tables , 8, 9.The test compounds 3a and 3b showed a concentration-dependent decrease in percentage cell viability with an increase in concentration in OECM 1 & UM- SCC 6 & HSC-3 cell lines. The test compound 3b produces maximum percentage inhibition of viability 63.19%, 62.49%, and 63.28% at 200μg/mL respectively, and minimum inhibition of cell viability 14.52%, 18.34%, and 15.6% at 6.25μg/mL concentration. Whereas 3a showed a maximum percentage inhibition of cell viability of 73.45%, 66.57%, and 76.31% at 200μg/mL and a minimum percentage inhibition of cell viability of 12.6%, 11.2%, and 14.35% at 6.5μg/ml  50 values were found to be 79.637 µg/mL (3a) and 160.051 µg/mL (3b) for OECM-1 followed by 94.235 µg/mL (3a) and 179.152 µg/mL (3b) for UM-SCC 6 with higher toxicity at HSC-3 cell line that was found to be 43.519 µg/mL (3a) and 92.391 µg/mL (3b) lethal even at a very low concentration. Among these, the test compounds, 3a produces more cytotoxicity towards the HSC-3 cell line followed by the OECM-1 cell line and the least being 3b compound at the UM-SCC6 cell line (160.051 μg/ml at 200 μg/ml concentration).MTT assayMTT assay was carried out to evaluate the inhibition concentration and measure the viable cells by determining the mitochondrial activity after 24 hours of specified compound concentration treatment with MTT assay expressed as mean ± SD Tables , 11, 12.Evidence-based studies showed cinnamic acid novel targeted derivatives exhibit anti-cancerous potential through HDAC inhibition by selective and less toxic molecular mechanisms. In the present study, two cinnamyl sulfonamide hydroxamate compounds  were assessed to explore the potential anti-cancerous activity of cinnamic acid derivatives. Pontiki et al.  observedA nitrous oxide scavenging assay was performed to evaluate the percentage inhibition of the antioxidant and free radical scavenging activity. Sarwar et al. observed good antioxidant activity with IC50 of 55.4 ± 0.21 μg/mL among Quercus incana roxb that increased with increasing concentration of the compound assay. Gryko et al.  in cinnaReducing potential was assessed by absorbance of different concentrations of compounds 3a and 3b at the given environment showed maximum absorbance (mean± SD) of 2.38 and 2.12 ± 0.001 and 0.006 respectively at 200μg/mL. Barre et al. in a study to evaluate the reduction potential and polypharmacy of cinnamic acid derivatives in type 2 diabetes mellitus (DM) patients observed derivatives such as chlorogenic acid, cinnamaldehyde, caffeic acid, and ferulic acid exhibited maximum reduction at lower concentrations . AdisakwA tryphan blue dye exclusion test was carried out to assess the number of sentinel cells present in the given OECM 1 & UM- SCC 6 & HSC-3 cell line suspension. Rodrigo et al. in a study employed cell lines derived for human oral SCC’s to assess the effect of raspberry-ethanol extract (RO-ET) activity on cellular characteristics using tryphan blue exclusion dye test observed cell proliferation without disorientation in the viability and cell suspension inhibition at 10, 50, and 100 μg/ml (> 97%) concentration that induces both apoptosis and differentiation . Ladke eMTT assay was carried out to evaluate the inhibition concentration and measure the viable cells by determining the mitochondrial activity after 24 hours of specified compound concentration treatment with MTT assay expressed as mean ± SD. In a study by Jayashree et al. 2-Quinolone Schiff's bases in a novel series of constituents  were produced. By using the MTT assay method, the active compounds were examined for their antiproliferative ability against lung cancer cell lines . The broIn the present study, LC 50 values of compound   and  for 24 hours were observed respectively thus suggesting a higher metabolic activity of compound 3a on the cell lines as an indicator of cell viability, proliferation, and cytotoxicity compared to compound 3b and high resistance potential displayed by these cell lines in response to the compounds 3a and 3b. Varadarajan et al. performed an in-vitro study to assess the anticancer property of cinnamic acid extracts using MTT assay -2,5-diphenyltetrazolium bromide) and observed anti-cancerous effect of 4-hydroxy-cinnamic acid and cinnamaldehyde through apoptosis by specific mitochondrial alteration in membrane potential and s-phase arrest in cell cycle .Reddy et al. in an assessment of synthesized cinnamyl derivatives  study showed cancer cell cytotoxicity with an effective increase in the apoptotic index and arrest in the G2/M phase in the cell cycle. Induction of p21 (Waf1/Cip1) expression, hyper-acetylation of H3 histidione, inhibition of induced MMP-2, and 9 expressions. In accordance with these results the present study also on the control treatment of compounds 3a and 3b with anisidine and toluidine revealed a statistically significant difference at G0/G1 phase and S phase. A significant decrease in cells in G0/G1 phase from 70.4% to 34.0 (%Gated) and an increase in cells in the S phase from 14.8% to 38.5% (gated %) was observed suggesting S phase arrest .Sova et al. demonstrated various biological effects of cinnamic acid derivatives (CAD) and cytotoxicity on HeLa, K562, Fem-x, and MCF-7 cell lines using MTT assay and showed significant cytotoxicity of the CAD compounds on the malignant cell lines . Cell stPlants are having effective medicinal value with reduced adverse effects. Future studies on the efficacy of the drug, mode of drug release and targeted drug delivery can be carried out using animal models. The extracted study molecule can also be compared with commercial anti-cancer agents for an effective drug delivery system. Exploring the targeted therapy and less harmful chemicals to treat cancer is a never-ending task. Recent rapid scientific advancements have improved our knowledge of cancer biology. As a result, a number of evolutionary targets have been found. The discovery of histone deacetylase as a new and potential target for cancer treatment. By inhibiting HDAC, cinnamic acid compounds demonstrate their anticancer effectiveness. Novel cinnamyl sulfonamide hydroxamate derivatives 3a and 3b were synthesized, characterized, and screened for their in vitro and in vivo anticancer potential. The compounds were subjected to anti-oxidant assays like ferric reducing power assay and NO scavenging assay. The test compounds 3a and 3b showed a concentration-dependent increase in the absorbance of Fe2+ in reducing power assay. MTT assay was carried out to evaluate the inhibition concentration and measure the viable cells, suggesting a higher metabolic activity of compound 3a on the cell lines as an indicator of cell viability, proliferation, and cytotoxicity compared to compound 3b and high resistance potential displayed by these cell lines in response to the compounds 3a and 3b.


While many factors have been implicated in breast cancer progression, effective treatments are still lacking. In recent years, it has become clear that posttranscriptional regulation plays a key role in the aberrant gene expression underlying malignancy and metastasis. For example, the mRNA modification N6-methyladenosine (m6A) is involved in numerous post-transcriptional regulation processes and has been implicated in many cancer types, including breast cancer. Despite intense study, even within a single type of cancer, there is little consensus, and often conflicting results, as to the role of m6A, suggesting other factors must influence the process. The goal of this study was to determine if the effects of m6A manipulation on proliferation and migration differed based on the stage of disease progression. Using the MCF10 model of breast cancer, we reduced m6A levels by targeting METTL3, the main cellular m6A RNA methyltransferase. Knocking down Mettl3 at different stages of breast cancer progression indeed shows unique effects at each stage. The early-stage breast cancer line showed a more proliferative phenotype with the knockdown of Mettl3 while the transformed breast cancer line showed a more migratory phenotype. Interestingly, the metastasized breast cancer cell line showed almost no effect on phenotype with the knockdown of Mettl3. Furthermore, transcriptome wide analysis revealed EMT as the probable pathway influencing the phenotypic changes. The results of this study may begin to address the controversy of m6A’s role in cancer and suggest that m6A may have a dynamic role in cancer that depends on the stage of progression. Despite this, there is still little understanding or effective treatments for advanced disease. The five-year survival rate for breast cancer drops 20 percent between stage 2 and 3 and an additional 50 percent between stage 3 and 4 . This deThe N6-methyladenosine (m6A) modification was discovered to be the most common mRNA modification nearly 50 years ago , 8. MettACH; underlined A is methylated) is only methylated in 10% of the mRNAs it is found in  supplemented with 5% Horse serum, 1% Penn/Strep, 0.2% Sodium Chloride, Insulin and Hydrocortisone, 0.07% Cholera toxin and 0.04% Epidermal Growth Factor.MCF10A, MCF10AT1 and MCF10CA1h cell lines were obtained from Karmanos Cancer center and maintained on 10 cm dishes  at 37°C, 5% CO2.2CRISPR/Cas9/sgRNA all-in-one construct co-expressing mCherry (Genecopoeia) was transfected using Lipofectamine 3000 (Thermo Fisher) according to manufacturer’s directions. Cells received either a control sgRNA construct (pCRISPR-CG01) or one targeting exon 1 of Mettl3 (HCP215070-CG12-3-10-a). Two rounds of transfection and Fluorescence Activated Cell Sorting (FACS) were used to isolate mCherry expressing cells which were then diluted to single cell for clonal selection. Western Blotting was used to confirm knockdown of Mettl3.2.3Trizol (Life Technologies) was used for all RNA extractions according to the manufacturer’s protocol. RNA for sequencing was further purified and treated with RNase-Free DNase I (Life Technologies) using PureLink RNA Mini Kit (Life Technologies). RNA purity and quantity was determined via NanoDrop 1000 (ThermoFisher Scientific).2.4Samples were sequenced by the Brody School of Medicine Integrative Genomics Core. RNA quality and concentration were verified by 4200 TapeStation evaluation (Agilent Technologies) and Qubit Fluorometric Quantitation (Thermo Fisher). Stranded cDNA libraries were prepared using TruSeq Stranded Total RNA prep and ligation with Ribo-Zero Plus kit (Illumina) in accordance with the manufacturer’s protocol. Paired-end sequencing (100 bp × 2) was performed on the NextSeq 2000 system (Illumina).For differential gene expression analysis, sequence reads were pseudo-aligned to the human hg38 reference transcriptome and transcript abundance quantified by using Kallisto (v.0.46.1). Differential gene expression analyses were achieved by using tximport (v1.20.0) and DESeq2 (v1.26.0) packages in R Studio (Build 351 with R v4.1.1) and further analyzed with GSEA and ShinyGo v0.77 . The RNA2.5For UPLC-MS/MS, purified PolyA+ RNA was digested to individual nucleosides and modified nucleosides were quantified as previously described .EpiQuik m6A RNA Methylation Quantification Kit (Colorimetric) was used to quantify relative m6A levels in 400 ng of total RNA according to the manufactures protocol.2.6Whole cell lysates prepared in whole cell extract buffer  were quantified with BCA Assay (Promega). Equal protein amounts were electrophoresed on a mini-PROTEAN any KD acrylamide gel (Bio-Rad Laboratories) and transferred to Hybond ECL nitrocellulose . The blot was blocked with 5% nonfat dry milk (LabScientific) in Tris buffered saline with 0.1% Tween 20 (TBST) for one hour at room temperature, followed by primary antibody in blocking buffer overnight at 4° C. After washing with TBST, blots were incubated with appropriate secondary HRP conjugated antibody , washed again with TBST, detected using Bio-Rad Clarity Western ECL Substrate, and imaged via Invitrogen Ibright FL1500 imaging system. A list of primary antibodies used can be found in 2.75 cells/well were plated in a 6 well plate. At 24, 48 or 72 hours from plating two wells were lifted with trypsin and counted via trypan blue and hemocytometer in duplicate. Remaining wells were fed with fresh media each day to prevent nutrient/additive deficiency during the time frame of the experiment and to allow for constant proliferation.Approximately 2.5x102.86, 1.5x106, and 1.0x106 cells/well were plated for the MCF10A, MCF10AT1 and MCF10CA1h cell line respectively in a 6 well plate. When the cells reach 95-100% confluency they were serum starved for 4 hours, scratched with a p20 pipette tip, washed with Dulbecco’s Phosphate Buffered Saline (DPBS) and fresh serum free media added. Pictures were taken at 0 and 24 hours and wound healing determined by measuring the change in distance between the cells on both sides of the scratch.Approximately 2.0x102.9Reverse transcription was performed on 1 μg of total RNA with the iScript cDNA synthesis kit (Bio-Rad Laboratories). Quantitative real-time PCR was performed using a Roche Lightcycler 96 with Fast Start Essential DNA Green (Roche Diagnostics Corporation) and primers from Integrated DNA Technologies, Inc. Primer efficiency was verified to be over 95% for all primer sets used. Quantification of mRNA was carried out via ΔΔCT analysis using 18S rRNA and the respective control condition for normalization. All real-time PCR primer sets were designed so the products would span at least one intron (>1kb when possible) to prevent detection of the pre-mRNA and/or DNA, and amplification of a single product was confirmed via melting curve analysis. Primer sequences can be found in 3This study aimed to characterize the unique role m6A has at differing stages of breast cancer progression utilizing the MCF10 model developed by the Karmanos Cancer Center at Wayne State University. The MCF10 cell lines were originally derived from benign proliferative breast tissue that spontaneously immortalized and are widely used to study cancer progression as well as EMT –34. We c3.1First, the m6A levels of each of the cell lines were determined via LC-MS of polyA selected, rRNA-depleted RNA . As showNext, the cell lines were characterized for the cancer-related phenotypes of proliferation and migration to verify that they modeled breast cancer progression as expected . ProlifeLastly, the state of EMT gene expression was examined to verify that the model also recapitulated known gene expression changes that accompany the epithelial to mesenchymal transition . Indeed,Overall, the MCF10 breast cancer progression model showed an increase in proliferation and migration between the MCF10A and MCF10AT1, and between the MCF10AT1 and MCF10CA1h cell lines along with a switch from epithelial to mesenchymal markers confirming the model as a valid representation of breast cancer progression.3.2To investigate the role of m6A in breast cancer progression, Mettl3 was knocked down via transient CRISPR/Cas9 transfection targeting exon 1 coupled with FACS sorting and clonal selection. Knockdown of Mettl3 was confirmed in each cell line via Western blotting and two candidates chosen for each line for use for this study  between the MCF10A and MCF10AT1. Similarly, between the MCF10A and MCF10CA1h cell lines 438 genes overlapped  Interestingly, the most overlap of significant gene changes was between the MCF10AT1 and MCF10CA1h with 667 genes overlapping  177 genes were found to be significantly altered in all three cell lines with the knockdown of Mettl3.Gene set enrichment analysis was then performed using ShinyGo . In the 3.4Next, m6A levels in total RNA of the cell lines with and without the knockdown of Mettl3 were quantified using a colorimetric, antibody-based quantification kit. With Mettl3 knockdown, a non-significant decrease in m6A was seen in the MCF10A cell line, , changes in expression of several EMT targets with the knockdown of Mettl3 were examined further by real-time PCR. In the MCF10A cell lines, which typically express high levels of epithelial markers, no change was observed in the mRNA expression of E-Cadherin, but N-Cadherin decreased about six-fold in both knockdowns while Vimentin decreased two to four-fold. Conversely, MMP2 saw a two and half fold increase in both knockdowns of Mettl3 in the MCF10A line , lowering m6A by knocking down Mettl3 caused the cells to become significantly more proliferative. This simultaneously caused the cells to become significantly less migratory as well. Similar results have been shown previously and support our findings . It has The transformed MCF10AT1 cells were developed by overexpression of an oncogenic, constitutively active version of HRas. Interestingly, these cells showed an opposite effect in response to the decrease in m6A in that, similar to other breast cancer studies, the proliferation rate significantly decreased, while the migration rate significantly increased , 27. ThiIn the malignant cell line MCF10CA1h, presumably large genomic changes have occurred during the serial mouse xenografts , 36, 37.We also investigated global changes in the transcriptome resulting from Mettl3 knock down in our breast cancer model to identify pathways that may be regulating the phenotypic changes observed. Interestingly, the cluster of genes that showed the most significant change in fold enrichment was EMT in both the MCF10A and MCF10AT1 cell lines. Consistent with the phenotypic changes, in general EMT genes went down in MCF10A and up in the MCF10AT1 cell lines  can be found in the article/Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.MD: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. BE: Data curation, Investigation, Writing – review & editing. CH: Data curation, Funding acquisition, Supervision, Writing – review & editing. KM: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Project administration, Supervision, Writing – review & editing.