Human Colon Carcinoma Cell Line Research Articles

Sorbate metal complexes as newer antibacterial, antibiofilm, and anticancer compounds

BackgroundThe ineffectiveness of treatments for infections caused by biofilm-producing pathogens and human carcinoma presents considerable challenges for global public health organizations. To tackle this issue, our study focused on exploring the potential of synthesizing new complexes of Co(II), Cu(II), Ni(II), and Zn(II) with sorbic acid to enhance its antibacterial, antibiofilm, and anticancer properties.MethodsFour novel complexes were synthesized as solid phases by reacting sorbic acid with Co(II), Cu(II), Ni(II), and Zn(II). These complexes were characterized by various technique, including infrared spectra, UV–Visible spectroscopy, proton nuclear magnetic resonance (1H NMR), and thermal analysis techniques, including thermogravimetry (TG).ResultsThe data acquired from all investigated chemical characterization methods confirmed the chemical structure of the sorbate metal complexes. These complexes exhibited antibacterial and antibiofilm properties against both Gram-positive and Gram-negative bacteria. Furthermore, these complexes enhanced the antibacterial effects of commonly used antibiotics, such as gentamicin and imipenem, with fractional inhibitory concentration (FIC) indices ≤ 0.5. Notably, the Cu(II) complex displayed the most potent antibacterial and antibiofilm activities, with minimum inhibitory concentration (MIC) values of 312.5 µg/mL and 625.0 µg/mL for Bacillus cereus and Escherichia coli, respectively. Additionally, in vitro assays using the methyl thiazolyl tetrazolium (MTT) method showed inhibitory effects on the growth of the human colon carcinoma cell line (HCT-116 cells) following treatment with the investigated metal complexes. The IC50 values for Co(II), Cu(II), Zn(II), and Ni(II) were 3230 µg/mL, 2110 µg/mL, 3730 µg/mL, and 2240 µg/mL, respectively.ConclusionOur findings offer potential for pharmaceutical companies to explore the development of novel combinations involving traditional antibiotics or anticancer drugs with sorbate copper complex.

Overcoming Resistance of Caco-2 Cells to 5-Fluorouracil through Diruthenium Complex Encapsulation in PMMA Nanoparticles.

Drug resistance, one of the main drawbacks in cancer chemotherapy, can be tackled by employing a combination of drugs that target different biological processes in the cell, enhancing the therapeutic efficacy. Herein, we report the synthesis and characterization of a new paddlewheel diruthenium complex that includes 5-fluorouracil (5-FU), a commonly used anticancer drug. This drug was functionalized with a carboxylate group to take advantage of the previously demonstrated release capacity of carboxylate ligands from the diruthenium core. The resulting hydrophobic complex, [Ru2Cl(DPhF)3(5-FUA)] (Ru-5-FUA) (DPhF = N,N'-diphenylformamidinate; 5-FUA = 5-fluorouracil-1-acetate) was subsequently entrapped in poly(methyl methacrylate) (PMMA) nanoparticles (PMMA@Ru-5-FUA) via a reprecipitation method to be transported in biological media. The optimized encapsulation procedure yielded particles with an average size of 81.2 nm, a PDI of 0.11, and a zeta potential of 29.2 mV. The cytotoxicity of the particles was tested in vitro using the human colon carcinoma cell line Caco-2. The IC50 (half maximal inhibitory concentration) of PMMA@Ru-5-FUA (6.08 μM) was just slightly lower than that found for the drug 5-FU (7.64 μM). Most importantly, while cells seemed to have developed drug resistance against 5-FU, PMMA@Ru-5-FUA showed an almost complete lethality at ∼30 μM. Conversely, an analogous diruthenium complex devoid of the 5-FU moiety, [Ru2Cl(DPhF)3(O2CCH3)] (PMMA@RuA), displayed a reduced cytotoxicity at equivalent concentrations. These findings highlight the effect of combining the anticancer properties of 5-FU with those of diruthenium species. This suggests that the distinct modes of action of the two chemical species are crucial for overcoming drug resistance.

Cell-cell contacts prevent t-BuOOH-triggered ferroptosis and cellular damage in vitro by regulation of intracellular calcium.

Tert-butyl hydroperoxide (t-BuOOH) is an organic hydroperoxide widely used as a model compound to induce oxidative stress. It leads to a plethora of cellular damage, including lipid peroxidation, DNA double-strand breaks (DNA DSBs), and breakdown of the mitochondrial membrane potential (MMP). We could show in several cell lines that t-BuOOH induces ferroptosis, triggered by iron-dependent lipid peroxidation. We have further revealed that not only t-BuOOH-mediated ferroptosis, but also DNA DSBs and loss of MMP are prevented by cell-cell contacts. The underlying mechanisms are not known. Here, we show in murine fibroblasts and a human colon carcinoma cell line that t-BuOOH (50 or 100µM, resp.) causes an increase in intracellular Ca2+, and that this increase is key to lipid peroxidation and ferroptosis, DNA DSB formation and dissipation of the MMP. We further demonstrate that cell-cell contacts prevent t-BuOOH-mediated raise in intracellular Ca2+. Hence, we provide novel insights into the mechanism of t-BuOOH-triggered cellular damage including ferroptosis and propose a model in which cell-cell contacts control intracellular Ca2+ levels to prevent lipid peroxidation, DNA DSB-formation and loss of MMP. Since Ca2+ is a central player of toxicity in response to oxidative stress and is involved in various cell death pathways, our observations suggest a broad protective function of cell-cell contacts against a variety of exogenous toxicants.

The Effect of Phenolic-Rich Extracts of Rubus fruticosus, R. ulmifolius and Morus nigra on Oxidative Stress and Caco-2 Inhibition Growth.

Currently, a clear interest has been given to berries due to their richness in active metabolites, including anthocyanins and non-coloured phenolics. Therefore, the main aim of the present work is to investigate the phenolic profile, antioxidant abilities, and antiproliferative effects on normal human dermal fibroblasts (NHDF) and human colon carcinoma cell line (Caco-2) cells of phenolic-rich extracts from three red fruits highly appreciated by consumers: two species of blackberries (Rubus fruticosus and Rubus ulmifolius) and one species of mulberry (Morus nigra). A total of 19 different phenolics were identified and quantified by HPLC-DAD-ESI/MSn and HPLC-DAD, respectively. Focusing on the biological potential of the phenolic-rich extracts, all of them revealed notable scavenging abilities. Concerning the antiproliferative properties, R. fruticosus presented a cytotoxic selectivity for Caco-2 cells compared to NHDF cells. To deeper explore the biological potential, combinations with positive controls (ascorbic acid and 5-fluorouracil) were also conducted. Finally, the obtained data are another piece of evidence that the combination of phenolic-rich extracts from natural plants with positive controls may reduce clinical therapy costs and the possible toxicity of chemical drugs.

Tailored multivalent peptide targeting the B-subunit pentamer of cholera toxin inhibits its intestinal toxicity by inducing aberrant transport of the toxin in cells

Cholera toxin (Ctx) is a major virulence factor produced by Vibrio cholerae that can cause gastrointestinal diseases, including severe watery diarrhea and dehydration, in humans. Ctx binds to target cells through multivalent interactions between its B-subunit pentamer and the receptor ganglioside GM1 present on the cell surface. Here, we identified a series of tetravalent peptides that specifically bind to the receptor-binding region of the B-subunit pentamer using affinity-based screening of multivalent random-peptide libraries. These tetravalent peptides efficiently inhibited not only the cell-elongation phenotype but also the elevated cAMP levels, both of which are induced by Ctx treatment in CHO cells or a human colon carcinoma cell line (Caco-2 cells), respectively. Importantly, one of these peptides, NRR-tet, which was highly efficient in these two activities, markedly inhibited fluid accumulation in the mouse ileum caused by the direct injection of Ctx. In consistent, NRR-tet reduced the extensive Ctx-induced damage of the intestinal villi. After NRR-tet bound to Ctx, the complex was incorporated into the cultured epithelial cells and accumulated in the recycling endosome, affecting the retrograde transport of Ctx from the endosome to the Golgi, which is an essential process for Ctx to exert its toxicity in cells. Thus, NRR-tet may be a novel type of therapeutic agent against cholera, which induces the aberrant transport of Ctx in the intestinal epithelial cells, detoxifying the toxin.

Bridged NHC-Pd(II) complexes: Synthesis, DFT calculations, molecular docking, and investigation of catalytic and biological activities

Six palladium(II) (3a-f) complexes of the type [Pd2(μ-Cl)2(NHC)] were prepared by transmetallation of the corresponding Ag-NHC and [PdCl2(CH3CN)2] complexes, and their structures were successfully characterised by 1H NMR, 13C NMR, HRMS, FTIR and elemental analysis. Density functional theory (DFT) calculations were also realised for the complexes. The prepared complexes were assessed for their catalytic activity in the C-H arylation of 2-isobuthylthiazole as well as for their biological activities. As results, these complexes were found to be potent catalysts in the creation of C5-arylated 2-isobuthylthiazole derivatives via C-H bond activation reaction. Furthermore, biological activity analysis revealed that complex 3a exhibits high cytotoxicity towards both human colon carcinoma cell lines (HCT-116) and hepatocellular carcinoma cell lines (HepG-2) with IC50 values of 4.2 and 9.3 µMmL−1, respectively. Complex 3b also showed antioxidant activity comparable to that of BHT through DPPH and ABTS assays. Both complexes 3d and 3f also showed significant inhibitory activity towards the AChE enzyme with IC50 values of 5.06 and 2.52 µMmL−1, respectively. Finally, all complexes showed excellent antiparasitic activity, with 3b exhibiting strong antileishmanial activity against both L. major promastigotes and amastigotes. The interaction between the most cytotoxic complexes and DNA, envisaged as a potential mechanism of toxicity, was explored by means of docking studies. In summary, these prepared complexes have the potential to serve as potent catalysts for the synthesis of arylated 2-isobutylthiazole and biologically active agents, paving the way for numerous prospects in the fields of medicinal chemistry and organic synthesis.

Uncovering the Power of HSCCC: Separation of Diastereomeric and Regioisomeric Styrylpyrones from the Stem Bark of Goniothalamus leiocarpus.

The plant Goniothalamus leiocarpus of the Annonaceae family is used as an alternative medicine in tropical regions. Applying high-speed counter current chromatography (HSCCC), eight new bioactive styrylpyrone isomers, including 6R,7S,8R,2'S-goniolactone B (1), 6S,7S,8S,2'S-goniolactone B (2), 6R,7R,8R,2'S-goniolactone B (3), 6R,7S,8S,2'S-goniolactone C (4), 6R,7S,8R,2'S-goniolactone C (5), 6S,7R,8S,2'S-goniolactone C (6), and two positional isomers, 6R,7R,8R,2'S-goniolactone G (7) and 6S,7R,8R,2'S-goniolactone G (8), were isolated from a chloroform fraction (2.1 g) of G. leiocarpus, which had a prominent spot by TLC analysis. The structures of the new compounds were elucidated by MS, NMR, IR, and UV spectra, and their absolute configurations were determined by Mosher's method, ECD, and X-ray diffraction analysis. The isolates are characteristic components found in plants of the genus Goniothalamus and consist of two structural moieties: a styrylpyrone and a dihydroflavone unit. The isolation of the eight new compounds demonstrates the effectiveness of HSCCC in separating the isomers of natural styrylpyrone. In a bioactivity assessment, compounds 1 and 6 exhibited cytotoxic effects against the human colon carcinoma cell lines LS513 and SW620 with IC50 values ranging from 1.6 to 3.9 μM. Compounds 1, 2, 7, and 8 showed significant synergistic activity against antibiotic-resistant Staphylococcus aureus strains.

In vitro 3D microfluidic peritoneal metastatic colorectal cancer model for testing different oxaliplatin-based HIPEC regimens.

Treatment of colorectal peritoneal metastases with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC) is still evolving. Conducting a randomized trial is challenging due to the high heterogeneity in the presentation of peritoneal disease and various surgical approaches. Biological research may facilitate more rapid translation of information into clinical practice. There is an emerging need for a preclinical model to improve HIPEC treatment protocols in terms of drug doses and treatment durations. The aim of the study is to design a tool that serves as an invitro three-dimensional (3D) microfluidic peritoneal metastatic colorectal cancer model to test the efficacy of different HIPEC treatments. We determined the effects of current therapy options using a 3D static disease model on human colon carcinoma cell lines (HCT 116) and transforming growth factor-β1 induced epithelial-to-mesenchymal transition (EMT) HCT 116 lines at 37 °C and 42 °C for 30, 60, and 120 min. We determined oxaliplatin's half maximal inhibitory concentrations in a 3D static culture by using viability assay. Clinical practices of HIPEC were applied in the developed model. EMT-induced HCT 116 cells were less sensitive to oxaliplatin treatment compared to non-induced cells. We observed increased cytotoxicity when increasing the temperature from 37 °C to 42 °C and extending the treatment duration from 30 to 120 min. We found that 200 mg/m2 oxaliplatin administered for 120 min is the most effective HIPEC treatment option within the framework of clinic applications. The tool map provide insights into creating more realistic pre-clinical tools that could be used for a patient-based drug screening.

Chili pepper extracts, capsaicin, and dihydrocapsaicin as potential anticancer agents targeting topoisomerases.

DNA topoisomerases regulate conformational changes in DNA topology during normal cell growth, such as replication, transcription, recombination, and repair, and may be targeted for anticancer drugs. A DNA topology assay was used to investigate DNA-damaging/protective activities of extracts from Habanero Red (HR), Habanero Maya Red (HMR), Trinidad Moruga Scorpion (TMS), Jalapeno (J), Serrano pepper (SP), Habanero Red Savina (HRS), Bhut Jolokia (BJ), and Jamaica Rosso (JR) peppers, demonstrating their inhibitory effect on the relaxation of pBR by Topo I. DNA topoisomerase II (Topo II) is proven therapeutic target of anticancer drugs. Complete inhibition of Topo II was observed for samples TMS, HR, and HMR. Extracts J and SP had the lowest capsaicin and dihydrocapsaicin content compared to other peppers. HR, HMR, TMS, J, S, HRS, BJ, JR extracts showed the anticancer effect, examined by MTS and xCell assay on the in vitro culture of human colon carcinoma cell line HCT116.

A64 HUMAN INTESINAL ORGANOIDS AS A MODEL FOR GASTROINTESTINAL FUNCTION AND TOXICITY SCREENING

Abstract Aims Drug-induced gastrointestinal (GI) toxicity is a common adverse event in clinical trials, with symptoms including diarrhea, ulceration, and inflammation resulting from impaired barrier function. Furthermore, the intestine is a key mediator of both drug absorption and metabolism. The human colon carcinoma cell line (Caco-2) is the most common model used in drug discovery, however, intestinal organoids provide a model that better recapitulates the self-renewal, cellular composition, and function of the intestinal epithelium.This study utilizes human intestinal organoids as a high-throughput model for evaluating intestinal function and toxicity in preclinical drug discovery. Methods To assess toxicity, intestinal organoids were treated with two GI-toxic drugs, gefitinib and colchicine, and post-treatment cell viability was compared to Caco-2 control cultures. To assess barrier function, organoid-derived monolayers cultured in Transwell® plates were treated with colchicine and sapitinib, and post-treatment permeability was evaluated using the fluorescent low-permeability markers, 4 kDa FITC-dextran and Lucifer yellow. Results Human intestinal organoid cultures exhibited 14-fold greater sensitivity to gefitinib (average organoid IC50 = 0.82 μM, n = 3; Caco-2 IC50 =11.4 μM, n = 2) and 2,238 times greater sensitivity to colchicine (average organoid IC50 = 0.028 μM, n = 3; Caco-2 IC50 = 62.89 μM, n = 6) compared to Caco-2 controls. This 96-well assay was reproducible, producing comparable IC50 concentrations in repeated experiments with cells from the same and different donors (n = 3 donors). Organoid-derived monolayers exhibited ampersand:003E 10-fold (n = 3) increase in permeability to both sapitinib and colchicine compared to Caco-2 cultures, which displayed minimal responses to either drug treatment. Finally, organoid-derived monolayer cultures in transwell® plates can be utilized to quantify drug transport and metabolism by the intestinal epithelium. Using ileal organoid-derived monolayers and Caco-2 cultures, we quantified the active transport of methotrexate by the efflux transporter BCRP, calculating an efflux ratio of 11.46 from the basal to apical chambers. We also quantified CYP3A4 activity of ileal monolayers relative to Caco-2 cultures. Ileal monolayers converted midazolam to hydroxymidazolam ampersand:003E 50-fold (n = 2) more than Caco-2 cultures, which displayed minimal CYP3A4 activity below the limit of detection. Together, these data demonstrate that human intestinal organoids are a valuable tool for high-throughput GI toxicity screening and for evaluating intestinal function in preclinical drug discovery. Conclusions Together, these data demonstrate that human intestinal organoids are a valuable tool for high-throughput GI toxicity screening and for evaluating intestinal function in preclinical drug discovery. Funding Agencies None

Surface Modification Strategies for Chrysin-Loaded Iron Oxide Nanoparticles to Boost Their Anti-Tumor Efficacy in Human Colon Carcinoma Cells.

Enhancing nanoparticles' anti-cancer capabilities as drug carriers requires the careful adjustment of formulation parameters, including loading efficiency, drug/carrier ratio, and synthesis method. Small adjustments to these parameters can significantly influence the drug-loading efficiency of nanoparticles. Our study explored how chitosan and polyethylene glycol (PEG) coatings affect the structural properties, drug-loading efficiency, and anti-cancer efficacy of Fe3O4 nanoparticles (NPs). The loading efficiency of the NPs was determined using FTIR spectrometry and XRD. The quantity of chrysin incorporated into the coated NPs was examined using UV-Vis spectrometry. The effect of the NPs on cell viability and apoptosis was determined by employing the HCT 116 human colon carcinoma cell line. We showed that a two-fold increase in drug concentration did not impact the loading efficiency of Fe3O4 NPs coated with PEG. However, there was a 33 Å difference in the crystallite sizes obtained from chitosan-coated Fe3O4 NPs and drug concentrations of 1:0.5 and 1:2, resulting in decreased system stability. In conclusion, PEG coating exhibited a higher loading efficiency of Fe3O4 NPs compared to chitosan, resulting in enhanced anti-tumor effects. Furthermore, variations in the loaded amount of chrysin did not impact the crystallinity of PEG-coated NPs, emphasizing the stability and regularity of the system.

Characterization of the effects of thymol derivatives on colorectal cancer spheroids.

Colorectal cancer (CRC) is one of the most commonly diagnosed malignancies with a high mortality rate. In the last few years, attention has been focused on substances of natural origin with anticancer activity. One such substance is thymol and its derivatives, which have been shown to have an antitumor effect also against CRC cells. In our study, we focused on determining the biological and antibacterial effects of thymol and thymol derivatives. Analyses were performed on a 3D model of human colon carcinoma cell lines (HCT-116 and HT-29) - spheroids. The cytotoxic (MTT assay) and genotoxic effect (comet assay) of thymol and derivatives: acetic acid thymol ester and thymol ß-D-glucoside were determined. ROS levels (ROS-Glo™ H2O2 Assay) and total antioxidant status (Randox TAS Assay) were also monitored. Last but not least, we also detected the effect of the derivatives using a disk diffusion assay and determined the number of colonies on the plates on selected bacteria such as Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum, Lacticaseibacillus paracasei, Lactobacillus brevis, Lactobacillus pentosus and Weizmannia coagulans. The derivatives did not show a significant inhibitory effect on the growth of LAB bacteria (lactic acid bacteria) in contrast to thymol. Overall, thymol derivatives are cytotoxic, genotoxic and increase ROS levels. Among the derivatives tested, acetic acid thymol ester (IC50 ~ 0.2 μg/ml) was more effective. The second derivative tested (thymol β-D-glucoside) was effective at higher concentrations than thymol. Our research confirmed that thymol derivatives have a toxic effect on the 3D model of intestinal tumor cells, while they do not have a toxic effect on selected intestinal bacteria. Thus, they could bring new significance to the prevention or treatment of CRC.

Gallic acid attenuates LPS-induced inflammation in Caco-2 cells by suppressing the activation of the NF-κB/MAPK signaling pathway.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease characterized by intestinal barrier dysfunction, inflammatory synergistic effects and excessive tissue injury. Gallic acid (GA) is renowned for its remarkable biological activity, encompassing anti-inflammatory and antioxidant properties. However, the underlying mechanisms by which GA protects against intestinal inflammation have not been fully elucidated. The aim of this study is to investigate the effect of GA on the inflammation of a lipopolysaccharide (LPS)-stimulated human colon carcinoma cell line (Caco-2) and on the intestinal barrier dysfunction, and explore the underlying molecular mechanism involved. Our findings demonstrate that 5 μg/mL GA restores the downregulation of the mRNA and protein levels of Claudin-1, Occludin, and ZO-1 and decreases the expressions of inflammatory factors such as IL-6, IL-1β and TNF-α induced by LPS. In addition, GA exhibits a protective effect by reducing the LPS-enhanced early and late apoptotic ratios, downregulating the mRNA levels of pro-apoptotic factors ( Bax, Bad, Caspase-3, Caspase-8, and Caspase-9), and upregulating the mRNA levels of anti-apoptotic factor Bcl-2 in Caco-2 cells. GA also reduces the levels of reactive oxygen species increased by LPS and restores the activity of antioxidant enzymes, namely, superoxide dismutase and catalase, as well as the level of glutathione. More importantly, GA exerts its anti-inflammatory effects by inhibiting the LPS-induced phosphorylation of key signaling molecules in the NF-κB/MAPK pathway, including p65, IκB-α, p38, JNK, and ERK, in Caco-2 cells. Overall, our findings show that GA increases the expressions of tight junction proteins, reduces cell apoptosis, relieves oxidative stress and suppresses the activation of the NF-κB/MAPK pathway to reduce LPS-induced intestinal inflammation in Caco-2 cells, indicating that GA has potential as a therapeutic agent for intestinal inflammation.

Chronic intermittent ethanol exposure-induced m6A modifications around mRNA stop codons of opioid receptor genes

ABSTRACT Chronic alcohol consumption may alter mRNA methylation and expression levels of genes related to addiction and reward in the brain, potentially contributing to alcohol tolerance and dependence. Neuron-like (SH-SY5Y) and non-neuronal (SW620) cells were utilized as models to examine chronic intermittent ethanol (CIE) exposure-induced global m6A RNA methylation changes, as well as m6A mRNA methylation changes around the stop codon of three opioid receptor genes (OPRM1, OPRD1, and OPRK1), which are known to regulate pain, reward, and addiction behaviours. CIE exposure for three weeks significantly increased global RNA methylation levels in both SH-SY5Y (t = 3.98, P = 0.007) and SW620 (t = 2.24, P = 0.067) cells. However, a 3-week CIE exposure resulted in hypomethylation around mRNA stop codon regions of OPRM1 and OPRD1 in both cell lines [OPRM1 (SH-SY5Y): t = -5.05, P = 0.0005; OPRM1 (SW620): t = -3.19, P = 0.013; OPRD1 (SH-SY5Y): t = -13.43, P < 0.00001; OPRD1 (SW620): t = -4.00, P = 0.003]. Additionally, mRNA expression levels of OPRM1, OPRD1, and OPRK1 were downregulated (corresponding to mRNA hypomethylation) in both SH-SY5Y and SW620 cells after a 3-week CIE exposure. The present study demonstrated that chronic ethanol exposure altered global RNA methylation levels, as well as mRNA methylation and expression levels of opioid receptor genes in both neuron-like and non-neuronal cells. Our findings suggest a potential epitranscriptomic mechanism by which chronic alcohol consumption remodels the expression of reward-related and alcohol responsive genes in the brain, thus increasing the risk of alcohol use disorder development. Abbreviations: OPRM1: the μ-opioid receptor; OPRD1: the δ-opioid receptor; OPRK1: the κ-opioid receptor; CIE: chronic intermittent ethanol exposure; CIE+WD: chronic intermittent ethanol exposure followed by a 24-hr withdrawal; SH-SY5Y: human neuroblastoma cell Line; SW620: human colon carcinoma cell line; RT-qPCR: reverse transcription followed by quantitative polymerase reaction; MazF-RT-qPCR: MazF digestion followed by RT-qPCR

Prediction of Methyl Orange (MO) Toxicity and Minimizing Its Pollution in Aquatic Environment by Activated Carbon Adsorption

Minimizing environmental pollution is an essential work of the official and scientific communities around the world, especially in water systems. In water, soluble dye works as a blockage in photosynthesis process because of its toxicity. One of these highly applicable dyes in industry is Methyl Orange (MO), documented with more than 10% released to water. Here, a new Iraqi try of converting environmental and health problems to solutions with high quantification was as done by using face tissue (Kleenex) as a carbon source. Primary in Silico testing of this anionic dye (Methyl Orange) was done based on online website confirmed MO dye is unsafe in several toxicological determinations such as foetus health (during pregnancy). Also, it is permeable material to skin, Blood- Brain system (BBB), and Human Colon Carcinoma cell line (CaCO2) compatible with Human Intestinal absorption (74.166%). In experimental section, low quality of face tissue (Kleenex) was subject to a high acidic medium (concentrated sulphuric acid), followed by addition of sodium carbonate to .increase activation of based carbon material with more structural pores yielding high removal efficiency and adsorption capacity ranged (88-98)% and (88-98) mg/g respectively. Qualitative and quantitative evaluations were based upon choosing two wavelengths in ultraviolet (272 nm) and visible (464 nm) regions. In this work, two removal steps were performed with the same adsorbent companied by multiple UV-Vis spectroscopic evaluation of several tested sections. Reviewing of published papers in MO removal presents the extraordinary performance of this prepared material towards using it as an excellent adsorbent of toxic material in aqueous solution.

Curcuminoid Chalcones: Synthesis and Biological Activity against the Human Colon Carcinoma (Caco-2) Cell Line.

There are many current scientific reports on the synthesis of various derivatives modelled on the structure of known small-molecular and natural bioactive compounds. Curcuminoid chalcones are an innovative class of compounds with significant therapeutic potential against various diseases and they perfectly fit into the current trends in the search for new biologically active substances. The aim of this study was to design and synthesise a series of curcuminoid chalcones. The objective of this scientific paper was to synthesise twelve curcuminoid chalcones and confirm their structures using spectral methods. Additionally, the biological activity of three of the synthesised compounds was evaluated using various assays, and their anticancer properties and toxicity were studied. The proposed derivatives were obtained via the Claisen-Schmidt reaction of selected acetophenones and aldehydes in various conditions using both classical methods: the solutions and solvent-free microwave (MW) or ultrasound (US) variants. The most optimal synthetic method for the selected curcuminoid chalcones was the classical Claisen-Schmidt condensation in an alkaline (NaOH) medium. Spectral methods were used to confirm the structures of the compounds. The resazurin reduction assay, caspase-3 activity assay, and RT-qPCR method were performed, followed by measurements of the intracellular reactive oxygen species (ROS) level and the lactate dehydrogenase (LDH) release level. Twelve designed curcuminoid chalcones were successfully synthesized and structurally confirmed by NMR, MS, and IR spectroscopy. Examination of the anticancer activity was carried out for the three most interesting chalcone products. The results suggested that compound 3a increased the metabolism and/or proliferation of the human colon carcinoma (Caco-2) cell line, while compounds 3b and 3f showed significant toxicity against the Caco-2 cell line. Overall, the preliminary results suggested that compound 3b exhibited the most favorable anticancer activity.

Effect of Bioreactor Cultures on the Proliferation and Biological Activity of Protocorm-like Bodies of Dendrobium loddigesii

Dendrobium loddigesii has long been used in traditional folk medicine. The purpose of this study was to optimize the culture conditions for its protocorm-like bodies (PLBs) and explore their biological activities. The use of an air-lift bioreactor demonstrated superior PLB proliferation compared to agitated and solid culture methods. The optimal inoculum quantity of 30 g/vessel, cultured for 28 days in the bioreactor, yielded the highest PLB biomass production. Analysis of PLB extracts revealed that the ethyl acetate (EtOAc) extract exhibited the highest levels of flavonoids and alkaloids, as well as potent antioxidant activity demonstrated by DPPH free radical scavenging assay and reducing power. Furthermore, the antiproliferative effects of the PLB extracts were assessed using MTT assays, and the EtOAc extract showed significant efficacy by reducing cell viability by over 60% in the human colon carcinoma cell line SW620 at the highest tested concentration (200 μg/mL). Mechanistic analysis revealed the downregulation of key regulatory apoptosis genes, including survivin, p53, caspase-3, and caspase-9. These results demonstrate the potential of the bioreactor culture method for the efficient production of D. loddigesii PLBs and the biological activities of the EtOAc extract, suggesting its therapeutic potential.

Swietenolide isolated from Swietenia macrophylla King in Hook seeds shows in vitro anti-colorectal cancer activity through inhibition of mouse double minute 2 (MDM2) homolog

Swietenia macrophylla King in Hook (SM) is known to have several medicinal properties. Chloroform extracts of SM seeds (SMCE) as well as two isolated limonoids swietenine (1) and swietenolide (2) showed significant in vitro anti-CRC activity in human colon carcinoma (HCT116) cell line. 2 (IC50 = 5.6 µM) was found to be two times more potent than 1 (IC50 = 10 µM). Both compounds showed anti-CRC activity through inhibition of the Mouse Double Minute 2 homolog (MDM2) of the MDM2-p53 pathway. The Selectivity Index (S.I.) of isolated compounds 1 and 2 for cancer cells were about 6.6 and 12.8 fold respectively which was significantly better than the S.I. of the extract (S.I. ∼1.5).

Development of analytical methods fort he quantitative analysis of the enzyme abundance and activity in the arachidonic acid cascade

The arachidonic acid (ARA) cascade is a key regulatory pathway where eicosanoids and other oxylipins are formed via the oxidation of polyunsaturated fatty acids. These oxylipins are involved in controlling physiological functions such as vascular tone, blood clotting and immune regulation. Changes in the oxylipin pattern can be caused by direct enzyme inhibition or modulated gene expression. In order to understand the effect mechanisms modulating the ARA cascade it is essential to not only investigate the metabolite, i.e. oxylipin, concentrations but also the abundances of the enzymes involved in their formation. For this reason, a quantitative targeted proteomics method for the ARA cascade was developed within this thesis.A detailed workflow for method development was established where enzyme/protein levels are measured via sequence‐specific, representative peptides chosen on the basis of several criteria. With liquid chromatography‐tandem mass spectrometry‐based analysis, the cyclooxygenase (COX) pathway of the ARA cascade was investigated in human colon carcinoma cell lines and primary macrophages where strong correlations were found between the oxylipins formed via the COX pathway and the COX‐2 abundance.The modulating effects of polyphenols on the 5‐lipoxygenase (LOX) pathway of the ARA cascade were investigated in cell‐free enzyme assays and human neutrophils. Resveratrol, its dimer ɛ‐viniferin and a resveratrol imine analogue directly inhibited the 5‐LOX with micromolar potencies and modulated the total oxylipin pattern in the neutrophils with remarkably different effects. Genistein did not act as a 5‐LOX inhibitor in the cell‐free assay but the modulated oxylipin pattern in the cells suggests a global cellular interference.The enzymes of the LOX pathway were included in the targeted proteomics method which together with an extended targeted oxylipin metabolomics platform enables a sensitive multi‐ omics analysis of the ARA cascade in a single sample. The comparison of the multiple reaction monitoring (MRM) detection and the MRM^‐based approach with an additional fragmentation step demonstrated that MRM is more favorable in targeted proteomics due to its higher sensitivity, greater linear range and higher multiplexing capacities. The analysis with the multi‐ omics approach showed that the 5 LOX protein is induced together with its product formation during the differentiation of human monocytic THP 1 cells to macrophages. M1‐like human primary macrophages were also characterized by the abundance of 5‐LOX and its activating protein (FLAP), while 15‐LOX and 15‐LOX‐2 dominated the protein pattern of the M2 like macrophages accompanied by high levels of the oxylipins formed via these enzymes. The developed methodology allows mechanistic investigation of a modulation of the ARA cascade as demonstrated by lipo‐poly‐saccharide stimulation as well as pharmaceutical treatment of the cells.Overall, with this thesis, a new quantitative proteomics strategy for the enzymes of the ARA cascade is established which can be easily extended to further proteins. The use of this technique in combination with targeted oxylipin metabolomics has provided new insights into the mechanisms involved in the modulation of the ARA cascade in human immune cells.

Abstract 2384: Quantitative phase imaging provides new insights into diverse morphologies in cell lines and their behavior in response to alternative cancer treatments

Abstract Quantitative phase imaging (QPI) through holographic time-lapse microscopy allows for non-invasive measurements of cell dry mass and the ability to track cells for long periods of time with minimal damage, making it most beneficial for research on cancer treatment. Research with this enhanced method of observation can help decipher various morphologies present in specific cell lines and show how each cell can respond differently to provided signals. In this assay, we studied N2a murine neuroblast differentiation in response to Retinoic acid, as well as the anticancerous activity of Saffron and UV in MDA-MB-468 breast carcinoma and HCT116 colon carcinoma cell lines. Cells were observed using Telight’s Q-Phase, a multimodal microscope that utilizes QPI holographic time-lapse technology with label-free monitoring. The cells were able to survive in optimum conditions for up to 96 hours without any appreciative loss of viability. With the emergence of phytochemicals being used as alternative cancer treatments, specific pigments in saffron have shown therapeutic effects against cancer tissues such as apoptosis and decreased carcinogen presence. A microanalysis of the effects of saffron on reducing the invasion potential, viability, and tumorigenicity of breast cancer cell lines was performed. QPI microscopy was performed on MDA-MB-468 human breast adenocarcinoma and HCT116 colon carcinoma cell lines treated with varying doses of saffron and ultraviolet radiation. Cell lines were observed under the microscope for 58-hour periods for changes in mass, movement, and replication. The quantitative data showed a reduction of projections from saffron treatment but did not show any significant reduction in cell movement over the 58-hours. These results were later confirmed through scratch wound and Boyden chamber assays. Similarly, when examining the process of N2a neuroblast differentiation, it was observed that cells do not move towards differentiation in a linear manner, but rather changed their direction and path several times before committing to their final phenotype. These types of observations are critical in understanding the biological processes behind differentiation and drug response and could not have been done without using the various quantitative and analysis tools provided by the QPI technology. Citation Format: Rida Khan, Meaghan McDonald, Albina Mikhaylova, Fatima K. Rehman. Quantitative phase imaging provides new insights into diverse morphologies in cell lines and their behavior in response to alternative cancer treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2384.