Human Colorectal Adenocarcinoma Cell Line Research Articles

Dentatacid A: An Unprecedented 2, 3-Seco-arbor-2, 3-dioic Triterpenoid from the Invasive Plant Euphorbia dentata, with Cytotoxicity Effect on Colon Cancer.

Euphorbia dentata Michx. is an invasive plant species in China, known for its toxicity and potential to reduce crop yields, posing numerous threats. To gain a deeper understanding of this invasive plant, phytochemical methods were employed to isolate 13 terpenoids (1-11, 19, 20) and 7 sterols (12-18) from the ethanol extract of E. dentata, identifying one new compound and 19 known compounds. Within spectroscopic methods such as NMR, HR-ESI-MS, and ECD, the structures and absolute configurations of these compounds were established. Among them, dentatacid A (11) possesses an unprecedented 2, 3-seco-arbor-2, 3-dioic skeleton within the potential biosynthetic pathway proposed. Dentatacid A also exhibited excellent anti-proliferative activity against the HT-29 (human colorectal adenocarcinoma) cell line, with an IC50 value of 2.64 ± 0.78 μM, which was further confirmed through network pharmacology and molecular docking. This study significantly expands the chemical diversity of E. dentata and offers new insights into the resource utilization and management of this invasive plant from the perspective of natural product discovery.

Immunomodulatory effects of live and UV-killed Bacillus subtilis natto on inflammatory response in human colorectal adenocarcinoma cell line in vitro.

Colorectal cancer (CRC) is a heterogeneous disease of the colon or rectum arising from adenoma precursors and serrated polyps. Recently, probiotics have been proposed as an effective and potential therapeutic approach for CRC prevention and treatment. Probiotics have been shown to alleviate inflammation by restoring the integrity of the mucosal barrier and impeding cancer progression. In this study, we aimed to investigate the immunomodulatory effects of live and UV-killed Bacillus subtilis natto on the inflammatory response in CRC. Caco-2 cells were exposed to various concentrations of live and UV- killed B. subtilis natto, and cell viability was assessed using MTT assay. Gene expression analysis of IL-10, TGF-β, TLR2 and TLR4 was performed using RT-qPCR. Our findings showed that both live and UV-killed B. subtilis natto caused significant reduction in inflammatory response by decreasing the gene expression of TLR2 and TLR4, and enhancing the gene expression of IL-10 and TGF-β in Caco-2 cells as compared to control group. The results of this study suggest that live and UV-killed B. subtilis natto may hold potential as a therapeutic supplement for modulating inflammation in CRC.

Cytotoxic Activity of Lepidium virginicum L. Methanolic Extract on Human Colorectal Cancer Cells, Caco-2, through p53-Mediated Apoptosis.

Colorectal cancer (CRC) is the third most common type of cancer worldwide. Its treatment options have had a limited impact on cancer remission prognosis. Therefore, there is an ongoing need to discover novel anti-cancer agents. Medicinal plants have gained recognition as a source of anti-cancer bioactive compounds. Recently, ethanolic extract of L. virginicum stems ameliorated dinitrobenzene sulfonic acid (DNBS)-induced colitis by modulating the intestinal immune response. However, no scientific study has demonstrated this potential cytotoxic impact on colon cancer cells. The objective of this study was to evaluate the cytotoxic effect of the methanolic extract of L. virginicum (ELv) on a human colorectal adenocarcinoma cell line (Caco-2) and to identify and quantify the phenolic compounds present in ELv extracts by liquid chromatography-mass spectrometry analysis. The cytotoxic activity was assessed using cell viability assays by reduction in the compound 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH). MTT and LDH assays revealed that the ELv decreases cell viability in the Caco-2 cell line in a concentration-dependent manner. Cell death was a result of DNA fragmentation and p53-mediated apoptosis. Eight phenolic acids and five flavonoids were identified and quantified in the stems. In conclusion, our findings demonstrate that the extract of L. virginicum possesses cytotoxic properties on Caco-2 cell line, suggesting that it could be a potential source of new drugs against CRC.

Effect of pasteurization and storage on the bioavailability of milk proteins during in vitro intestinal digestion and Caco-2 cell transport

This study fills a specific knowledge gap by comprehensively investigating the intestinal digestive dynamics and transport kinetics of pasteurized milk proteins during cold storage, utilizing dynamic in vitro digestion models, human colorectal adenocarcinoma cell line (Caco-2) transport simulations, peptideomics and amino acid analysis. The content of free amino acids during intestinal digestion and their transport capacity (excluding lysine) were highest in pasteurized milk stored for 7 days (PAST-D7), intermediate in pasteurized milk stored for 0 days (PAST-D0) and lowest in raw milk (RM). Conversely, the quantity and intensity of bioactive peptides during digestion decreased with pasteurization and storage. Additionally, pasteurization and storage reduced the intensity of bioactive peptides transported through Caco-2 cells and the types and intensity of potential allergenic peptides. These findings indicate that while pasteurization and storage may diminish some physiological benefits of milk, they may also enhance protein bioavailability and reduce allergenicity.

Synthesis of new phenothiazine derivatives: Molecular docking, assessment of cytotoxic activity and oxidant-antioxidant properties on PCS-201-012, HT-29, and SH-SY5Y cell lines.

Phenothiazine (PTZ) derivatives have been acknowledged as versatile compounds with significant implications across various areas of medicine, particularly, in cancer research. The cytotoxic effects of synthesized compounds on both normal and cancerous cells, along with their oxidant-antioxidant properties, are pivotal factors in cancer treatment strategies. In the current study, eight new PTZ derivatives were synthesized and the compounds' cytotoxic activities were assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay while the oxidant-antioxidant properties were evaluated by oxidative stress index (OSI) calculation in SH-SY5Y (a human neuroblastoma cell line), HT-29 (a human colorectal adenocarcinoma cell line), and PCS-201-012 (a human primary dermal fibroblast cell line) cells. Consequently, the half-maximal inhibitory concentration (IC50) values of compound 3a were determined to be 218.72, 202.85, and 227.86 μM while the IC50 values of compound 3b were defined to be 227.42, 199.27, and 250.11 μM in PCS-201-012, HT-29, and SH-SY5Y cells, respectively. Additionally, it was determined that the synthesized compounds demonstrated the lowest OSI in PCS-201-012 cells as compared to the other cell lines.

Citrus Pomace as a Source of Plant Complexes to Be Used in the Nutraceutical Field of Intestinal Inflammation.

This study aims to recover the main by-product of Citrus fruits processing, the raw pomace, known also as pastazzo, to produce plant complexes to be used in the treatment of inflammatory bowel disease (IBD). Food-grade extracts from orange (OE) and lemon (LE) pomace were obtained by ultrasound-assisted maceration. After a preliminary phytochemical and biological screening by in vitro assays, primary and secondary metabolites were characterized by proton nuclear magnetic resonance (1H-NMR) and liquid chromatography coupled to diode array detection and electrospray ionization mass spectrometry (LC-DAD-ESI-MS) analyses. The intestinal bioaccessibility and antioxidant and anti-inflammatory properties were investigated by in vitro simulated gastro-intestinal digestion followed by treatments on a lipopolysaccharide (LPS)-stimulated human colorectal adenocarcinoma cell line (Caco-2). The tight junctions-associated structural proteins (ZO-1, Claudin-1, and Occludin), transepithelial electrical resistance (TEER), reactive oxygen species (ROS)-levels, expression of some key antioxidant (CAT, NRF2 and SOD2) and inflammatory (IL-1β, IL-6, TNF-α, IL-8) genes, and pNFkB p65 nuclear translocation, were evaluated. The OE and LE digesta, which did not show any significant difference in terms of phytochemical profile, showed significant effects in protecting against the LPS-induced intestinal barrier damage, oxidative stress and inflammatory response. In conclusion, both OE and LE emerged as potential candidates for further preclinical studies on in vivo IBD models.

Short Caco-2 model for evaluation of drug permeability: A sodium valerate-assisted approach

The human colorectal adenocarcinoma cell line Caco-2, widely used for studying intestinal drug permeability, is typically grown on permeable filter supports and matures in 21 days with frequent media changes. The process is labor-intensive, prone to contamination, and has low throughput, contributing to the overall high utilization cost. Efforts to establish a low-cost, high-throughput, and short-duration model have encountered obstacles, such as weaker tight junctions causing monolayer leaks, incomplete differentiation resulting in low transporter expression, intricate and challenging protocols, and cytotoxicity, limiting the usability. Hence, this study aimed to develop a low-cost, efficient, and short-duration model by addressing the aforementioned concerns by customizing the media and finding a safe differentiation inducer. We generated a new rapid model using sodium valerate, which demonstrated sufficient transporter activity, improved monolayer integrity, and higher levels of differentiation markers than the 21-day model. Furthermore, this model exhibited consistent and reliable results when used to evaluate drug permeability over multiple days of repeated use. This study demonstrates the potential of a sodium valerate-assisted abbreviated model for drug permeability assessment with economic and practical advantages.

A Potential Anticancer Property of Fermented and Unfermented Soybeans in Human Colorectal Adenocarcinoma Cell Line

A Potential Anticancer Property of Fermented and Unfermented Soybeans in Human Colorectal Adenocarcinoma Cell Line

Novel tetraaza macrocyclic Schiff base complexes of bivalent zinc: microwave-assisted green synthesis, spectroscopic characterization, density functional theory calculations, molecular docking studies, in vitro antimicrobial and anticancer activities.

In the present manuscript, novel macrocyclic Schiff base complexes [Zn(N4MacL1)Cl2-Zn(N4MacL3)Cl2] were synthesized by the reaction of ZnCl2 and macrocyclic ligands (N4MacL1-N4MacL3) derived from diketone and diamines under microwave irradiation method and conventional method. The structures of the obtained complexes were identified by various spectrometric methods such as Fourier transformation infra-red (FT-IR), nuclear magnetic resonance (NMR), high-resolution mass spectrometry (HR-MS), powder X-ray diffraction, molar conductivity, and UV-vis. The structures of the synthesized compounds were optimized by using the def2-TZV/J and def2-SVP/J Coulomb fitting basis sets at B3LYP level in density functional theory (DFT) calculations. The macrocyclic Schiff base complexes exhibited higher activities against Gram-positive bacteria (Staphylococcus aureus and Bacillus cereus), Gram-negative bacteria (Escherichia coli and Xanthomonas campestris), and fungal strains (Fusarium oxysporum and Candida albicans) in comparison to macrocyclic Schiff base ligands. Furthermore, the newly synthesized macrocyclic compounds were assessed for their anticancer activity against three cell lines: A549 (human alveolar adenocarcinoma epithelial cell line), HT-29 (human colorectal adenocarcinoma cell line), and MCF-7 (human breast adenocarcinoma cell line) using the MTT assay. The obtained results showed that the macrocyclic complex [Zn(N4MacL3)Cl2] displayed the highest cytotoxic activity (2.23 ± 0.25µM, 6.53 ± 0.28µM, and 7.40 ± 0.45µM for A549, HT-29, and MCF-7 cancer cell lines, respectively). Additionally, molecular docking investigations were conducted to elucidate potential molecular interactions between the synthesized macrocyclic compounds and target proteins. The results revealed a consistent agreement between the docking calculations and the experimental data.

Quantitative Analysis of Isopimpinellin from Ammi majus L. Fruits and Evaluation of Its Biological Effect on Selected Human Tumor Cells.

Ammi majus L. (Apiaceae) is a medicinal plant with a well-documented history in phytotherapy. The aim of the present work was to isolate isopimpinellin (5,8-methoxypsoralen; IsoP) from the fruit of this plant and evaluate its biological activity against selected tumor cell lines. The methanol extract obtained with the use of an accelerated solvent extraction (ASE) method was the most suitable for the quantitative analysis of coumarins in the A. majus fruit matrix. The coumarin content was estimated by RP-HPLC/DAD, and the amount of IsoP was found to be 404.14 mg/100 g dry wt., constituting 24.56% of the total coumarin fraction (1.65 g/100 g). This, along with the presence of xanthotoxin (368.04 mg/100 g, 22.36%) and bergapten (253.05 mg/100 g, 15.38%), confirmed A. majus fruits as an excellent source of these compounds. IsoP was isolated (99.8% purity) by combined liquid chromatography/centrifugal partition chromatography (LC/CPC) and tested for the first time on its antiproliferative activity against human colorectal adenocarcinoma (HT29, SW620), osteosarcoma (Saos-2, HOS), and multiple myeloma (RPMI8226, U266) cell lines. MTT assay results (96 h incubation) demonstrated a dose- and cell line-dependent decrease in cell proliferation/viability, with the strongest effect of IsoP against the Saos-2 cell line (IC50; 42.59 µM), medium effect against U266, HT-29, and RPMI8226 (IC50 = 84.14, 95.53, and 105.0 µM, respectively), and very weak activity against invasive HOS (IC50; 321.6 µM) and SW620 (IC50; 711.30 µM) cells, as well as normal human skin fibroblasts (HSFs), with IC50; 410.7 µM. The mechanistic study on the Saos-2 cell line showed that IsoP was able to reduce DNA synthesis and trigger apoptosis via caspase-3 activation. In general, IsoP was found to have more potency towards cancerous cells (except for HOS and SW620) than against healthy cells. The Selective Index (SI) was determined, underlining the higher selectivity of IsoP towards cancer cells compared to healthy cells (SI = 9.62 against Saos-2). All these results suggest that IsoP might be a promising molecule in the chemo-prevention and treatment of primary osteosarcoma.

PARP inhibitor synthetic lethality in ATM biallelic mutant cancer cell lines is associated with BRCA1/2 and RAD51 downregulation.

Ataxia telangiectasia-mutated (ATM) kinase is a central regulator of the DNA damage response (DDR) signaling pathway, and its function is critical for the maintenance of genomic stability in cells that coordinate a network of cellular processes, including DNA replication, DNA repair, and cell cycle progression. ATM is frequently mutated in human cancers, and approximately 3% of lung cancers have biallelic mutations in ATM, i.e., including 3.5% of lung adenocarcinomas (LUAD) and 1.4% of lung squamous cell carcinomas (LUSC). We investigated the potential of targeting the DDR pathway in lung cancer as a potential therapeutic approach. In this context, we examined whether ATM loss is synthetically lethal with niraparib monotherapy. This exploration involved the use of hATM knockout (KO) isogenic cell lines containing hATM homozygous (-/-) and heterozygous (+/-) generated via CRISPR/Cas9 gene knockout technology in DLD-1, a human colorectal adenocarcinoma cell line. Subsequently, we extended our investigation to non-small cell lung cancer (NSCLC) patient derived xenograft (PDX) models for further validation of poly ADP-ribose polymerase inhibitor (PARPi) synthetic lethality in ATM mutant NSCLC models. Here, we demonstared that biallelic hATM deletion (-/-) in DLD-1 impairs homologous recombination (HR) repair function and sensitizes cells to the PARPi, niraparib. Niraparib also caused significant tumor regression in one-third of the NSCLC PDX models harboring deleterious biallelic ATM mutations. Loss of hATM (-/-) was concomitantly associated with low BRCA1 and BRCA2 protein expression in both the hATM (-/-) DLD-1 cell line and PARPi-sensitive ATM mutant NSCLC PDX models, suggesting a downstream effect on the impairment of HR-mediated DNA checkpoint signaling. Further analysis revealed that loss of ATM led to inhibition of phosphorylation of MRN (Mre11-Rad50-NBS1) complex proteins, which are required for ATM-mediated downstream phosphorylation of p53, BRCA1, and CHK2. Taken together, our findings highlight that the synthetic lethality of niraparib in ATM-deficient tumors can be regulated through a subsequent effect on the modulation of BRCA1/2 expression and its effect on HR function.

Abstract 3178: Vitamin B1 derivative, fursultiamine, prevents colon cancer growth

Abstract Colon cancer is the second deadliest type of cancer in the United States, with surgical resection being the primary treatment modality for localized early-stage colon cancer. Still, more treatment and prevention options are needed to help improve the outcomes for colon cancer as they are currently limited to invasive procedures. One promising option is fursultiamine, a disulfide lipid-soluble vitamin B1 derivative, which has anti-oxidative and anti-inflammatory activity that could aid in preventing colon cancer. We hypothesize that supplementation of fursultiamine could be an effective chemopreventive treatment to control the growth and spread of colon carcinogenesis. Human colorectal adenocarcinoma (SW480) cell lines were incubated with complete media (CM) or human recombinant endothelial growth factor (EGF 20 ng/ml) in the absence and presence of fursultiamine. The cell viability was examined by MTT assay in a concentration- (0-150 uM) and time (24 and 48 h) - dependent manner. Our results indicate that fursultiamine prevents SW480 cell death in a dose-dependent manner. Apoptosis was detected by using Annexin-V staining and live and death cell assay. Our results suggest that fursultiamine prevents SW480 cell proliferation by inducing apoptotic cell death. The production of reactive oxygen species (ROS) and activation of caspase-3 were examined by specific assay kits. Treatment with fursultiamine resulted in the decreased production of ROS and activation of caspase-3. The expression of anti-apoptotic, pro-apoptotic, and pro-inflammatory factors was analyzed using antibody arrays. The results demonstrated that treatment with fursultiamine increased the expression of pro-apoptotic factors such as Bad, Bax, cleaved caspase 3, Fas/CD95, and FADD. Further, fursultiamine increased expression of the tumor suppressor genes p53, p27, and p21. We next planned to examine this vitamin B1 derivative's effect in preventing cancer growth in nude mice xenograft models. In conclusion, our in vitro results indicate that fursultiamine, a vitamin B1 derivative, prevents colon cancer growth through increased regulation of pro-apoptotic pathways and tumor suppressor proteins and thus has the potential for further development as a chemopreventative agent. Citation Format: Sudeep Poludasu, Christian Pompoco, Kota Ramana. Vitamin B1 derivative, fursultiamine, prevents colon cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3178.

Enhancing antibacterial efficacy and accelerating infectious wound healing in rats using biogenic metal nanoparticles from marine Bacillus subtilis

IntroductionMicroorganisms originating from the marine environment, such as bacteria, fungi, and algae, are deliberately employed in the production of nanoparticles on account of the wide array of bioactive compounds they produce.MethodsCell-free aqueous extracts of marine Bacillus subtilis (CBPPR1) were used to synthesise AuNPs (CBPPR1AuNPs) and AgNPs (CBPPR1AgNPs). Zetasizer Nano ZS (Malvern Instruments) zeta size and zeta potential, field emission and transmission scanning electron microscopy (FE-SEM and HR-TEM), UV-visible (UV-Vis), X-ray diffraction (XRD), Fourier transform infrared (FT-IR), and EDAX were used to characterize biogenically synthesized nanoparticles (NPs). Their antibacterial activities against Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus were investigated. The anticancer efficacy of CBPPR1Au and AgNPs was investigated in human colorectal adenocarcinoma cell lines (HT-29, HT-116). CBPPR1AgNPs formulation was studied in vitro and in-vivo rat models. The assessment focused on its efficacy in wound healing and antibacterial capabilities, comparing them against a commercial product. To determine the effectiveness of CBPPR1AgNPs in wound healing, a cutaneous wound model was employed, which included infection with S. aureus.Results and discusionCBPPR1Au and AgNPs significantly inhibited the growth of S aureus at MIC of 125 µg (CBPPR1AuNPs) and 62.5 µg (CBPPR1AgNPs) respectively. FE-SEM and HR-TEM observations confirmed that NPs caused bacterial cell leakage, damage, and shrinkage. Cancer cell viability was reduced upon treatment with increasing concentrations of CBPPR1Au and AgNPs, and apoptosis was increased in cells treated with CBPPR1Au and AgNPs relative to untreated cells (p < 0.001). CBPPR1Au and AgNPs showed significant cytotoxic activity against HT-29 (15.5 M) and HT-116 (62.5 M) cells. In-vivo experiments on rats showed minimal pus formation in groups CBPPR1AgNPs (62.5 µg/ml) G2, CBPPR1AgNPs (125 µg/ml) G3, and silver sulfadiazine G4, indicating the effective control of infections. CBPPR1AgNPs-treated wounds showed complete closure, whereas untreated G1 wounds remained unhealed. Histopathological analysis showed no adverse effects of CBPPR1AgNPs on kidneys and livers of rats. These findings suggest that CBPPR1AgNPs play a pivotal role in wound healing because of their potent antibacterial properties.

The inhibition effects of Lentilactobacillus buchneri-derived membrane vesicles on AGS and HT-29 cancer cells by inducing cell apoptosis

In recent years, probiotics and their derivatives have been recognized as important therapeutic agents in the fight against cancer. Therefore, this study aimed to investigate the anticancer effects of membrane vesicles (MVs) from Lentilactobacillus buchneri strain HBUM07105 probiotic isolated from conventional and unprocessed yogurt in Arak province, Iran, against gastric and colon cancer cell lines. The MVs were prepared from the cell-free supernatant (CFS) of L. buchneri and characterized using field-emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) and SPS-PAGE techniques. The anticancer activity of MVs was evaluated using MTT, flow cytometry, qRT-PCR techniques, and a scratch assay. The study investigated the anti-adenocarcinoma effect of MVs isolated from L. buchneri on a human gastric adenocarcinoma cell line (AGS) and a human colorectal adenocarcinoma cell line (HT-29) at 24, 48, and 72-h time intervals. The results demonstrated that all prepared concentrations (12.5, 25, 50, 100, and 200 µg/mL) of MVs reduced the viability of both types of human adenocarcinoma cells after 24, 48, and 72 h of treatment. The analysis of the apoptosis results revealed that the percentage of AGS and HT-29 cancer cells in the early and late stages of apoptosis was significantly higher after 24, 48, and 72 h of treatment compared to the untreated cancer cells. After treating both AGS and HT-29 cells with the MVs, the cells were arrested in the G0/G1 phase. These microvesicles demonstrate apoptotic activity by increasing the expression of pro-apoptotic genes (BAX, CASP3, and CASP9). According to the scratch test, MVs can significantly decrease the migration of HT-29 and AGS cancer cells after 24, 48, and 72 h of incubation compared to the control groups. The MVs of L. buchneri can also be considered a potential option for inhibiting cancer cell activities.

Transcription Silencing and CpGs Hypermethylation as Therapeutic Gene Editing in Clinical Colorectal Adenocarcinoma Repression.

Colorectal cancer is the most common cancer in oncopathology, with an increasing incidence among the elderly during the last decade. Various genetic and environmental factors play important roles in the emergence of colorectal adenocarcinoma. Non-coding RNAs, approximately 20-22 nucleotides, are transcribed irregularly in many cancer cells and play a critical role in many metabolic pathways in clinical cancer cases. DNA methylation is a critical epigenetic alteration that controls gene expression. In the current study, transcriptional silencing and CpG hypermethylation were developed as a therapeutic gene editing strategy for the clinical repression of colorectal adenocarcinoma. A human colorectal adenocarcinoma cell line (Caco2) and a normal lung fibroblast cell line (Wi38) were utilized as the paradigms in this research to examine the effect of mir155 molecule transfection and CpGs-island (CGI) methylation. Cell counting was achieved using six-well and 24-well plates before transfection using a hemocytometer. The two cell lines were transfected with the mir155 agomir and antagomir molecules. The transfection efficiency, cell viability, cell IC50, and target gene expression were measured, and CGIs-methylation was achieved by bisulfate conversion. The outcomes revealed the downregulation of oncogenes (AKT1 and VCAM1 genes as cancer-associated genes) and the upregulation of tumor suppressor genes (TSGs, Tp53 and KEAP1). In addition, CpG-islands methylation showed significant blocking of the oncogene promoter regions, and the switch on of TSG promoter regions was continuous. miRNA-CGI-methylation led to the regression of Caco2 cell proliferation, suggesting the potential use of RNA silencing and DNA methylation in targeted gene therapy for colorectal cancer.

Novel 2-(hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide based thiosemicarbazides as potent and selective inhibitors of tumor-associated human carbonic anhydrase IX and XII: Synthesis, cytotoxicity, and molecular modelling studies

In the pursuit of discovering new selective carbonic anhydrase (CA, EC 4.2.1.1) inhibitors, a small collection of novel thiosemicarbazides (5a-5t) were designed and synthesized starting from 2-(hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide which was evaluated as a potent inhibitor of different CA isoforms in a previous study. The newly synthesized compounds were examined against four human carbonic anhydrases (hCA), namely transmembrane tumor-related hCA IX/XII and cytosolic widespread off-targets hCA I/II. In enzyme inhibition assays, all nineteen compounds display up to ∼340-fold selectivity for hCA IX/XII over off-target isoforms hCA I/II. Four compounds have enzyme inhibition values (Ki) lower than 10 nM against tumor-associated isoforms hCA IX/XII including two compounds in the subnanomolar range (5r and 5s; hCA XII; Ki: 0.69 and 0.87 nM). The potential binding interactions of the most potent compounds against hCA IX and XII, compounds 5s and 5r, respectively, were investigated using ensemble docking and molecular dynamics studies. Cell viability assays using human colorectal adenocarcinoma cell line HT-29 and healthy skin fibroblasts CCD-86Sk show that compound 5e selectively inhibits HT-29 cancer cell proliferation (IC50: 53.32 ± 7.74 µM for HT-29; IC50: 74.64 ± 14.15 µM for CCD-986Sk). Finally, Western blot assays show that compounds 5e and 5r significantly reduce the expression of hCA XII in HT-29 cells. Moreover, 5e shows better cytotoxic activity in hypoxia compared to normoxic conditions. Altogether, the newly designed compounds show stronger inhibition of the tumor-associated hCA IX and XII isoforms and several tested compounds show selective cytotoxicity as well as downregulation of hCA XII expression.

Characterizing differential efficacy and phenotypic response to proteasome and survivin inhibitors in colorectal cancers using a high throughput organoid assay.

153 Background: Conventional monolayer cell cultures and xenograft models, while useful and economical in early drug discovery, cannot predict clinical efficacy. Further, preclinical screening assays that rely on differential metabolic activity between separate control and treated wells are incapable of capturing phenotypic response and could overstate efficacy for cells with high rates of proliferation. Consequently, over 95% of anticancer agents that show efficacy in preclinical studies, fail in clinical trials. Recently, patient-derived organoid (PDO) models have been utilized in developing platforms to predict clinical efficacy of preclinical formulations. If successful, such predictive ex vivo technologies could revolutionize cancer treatment by reducing cost and time-to-market for new, more effective therapeutics. Objective: Characterize a novel bioprinted organoid tumor (BOT) high-throughput screening ex vivo platform for drug response prediction (DRP) with known proteosome and survivn inhibitors in colorectal cancer. Methods: Bioink for 3D printing BOTs was prepared with HT-29 cells, an established NCI-60 human colorectal adenocarcinoma cell line with known sensitivity to proteosome and survivin inhibitors. Bioink was deposited layer-by-layer on multiple substrates, in various geometrical configurations, and cured in stages to allow cells and matrix to self-assemble with limited degrees of freedom. BOTs were screened 24h and 48h after printing with proteosome inhibitor Bortezomib and survivin inhibitor YM-155. BOTs were evaluated 48h and 72h after treatment using immunofluorescence live/dead assay. Morphological phenotypic changes resulting from treatment were also recorded. Results: Proteasome and survivin inhibitors have been reported to inhibit proliferation and induce cell death in colorectal cancer cells. A dose dependent response was observed for both agents in our novel BOT HTS thereby validating the platform. In addition, characteristic self-assembly of HT-29 cells was observed to be disrupted at effective doses and at certain concentrations below the effective dose. Traditional ATP assays are incapable of recording such phenotypic modulation. Further, a higher proliferation profile was observed in untreated BOTs suggesting that use of independent control wells in traditional assays could overstate efficacy of treatment. Conclusions: Functional high-throughput ex vivo DRP technologies have the potential to transform cancer treatment – from bench to bedside – along the drug discovery to market roadmap for much needed novel anticancer agents.

Lactiplantibacillus plantarum MIUG BL21 paraprobiotics: Evidences on inactivation kinetics and their potential as cytocompatible and antitumor alternatives

Two new -biotics concepts, such as paraprobiotics and postbiotics were introduced, with beneficial effects beyond the viability of probiotic. In this study, the effect of individual (thermal, ohmic heating, high pressure, and ultrasound) and combined (ohmic, high pressure and ultrasound in combination with heating) treatments on the inactivation kinetics of Lactiplantibacillus plantarum was investigated. Different inactivation rates were obtained, up to 8.18 after 10 min at 90 °C, 2.07 after 15 min at a voltage gradient of 20 V/cm, 6.62 after 10 min at 600 MPa and 3.6 after ultrasound treatment for 10 min at 100 % amplitude. The experimental data were fitted to Weibullian model proposed by Peleg, allowing to estimate the inactivation rate coefficient (b) and the shape of the inactivation curves (n). At lower concentration, the samples showed both cytocompatibility and antiproliferative effect, stimulating the cell proliferation on both murine fibroblast and human colorectal adenocarcinoma cell lines.

HucMSC-Ex alleviates DSS-induced colitis in mice by decreasing mast cell activation via the IL-33/ST2 axis.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease that poses challenges in terms of treatment. The precise mechanism underlying the role of human umbilical cord mesenchymal stem cell-derived exosome (HucMSC-Ex) in the inflammatory repair process of IBD remains elusive. Mucosal mast cells accumulate within the intestinal tract and exert regulatory functions in IBD, thus presenting a novel target for addressing this intestinal disease. A mouse model of Dextran Sulfate Sodium (DSS)-induced colitis was established and hucMSC-Ex were administered to investigate their impact on the regulation of intestinal mast cells. An in vitro co-culture model using the human clonal colorectal adenocarcinoma cell line (Caco-2) and human mast cell line (LAD2) was also established for further exploration of the effect of hucMSC-Ex. We observed the accumulation of mast cells in the intestines of patients with IBD as well as mice. In colitis mice, there was an upregulation of mast cell-related tryptase, interleukin-33 (IL-33), and suppression of tumorigenicity 2 receptor (ST2 or IL1RL1), and the function of the intestinal mucosal barrier related to intestinal tight junction protein was weakened. HucMSC-Ex treatment significantly reduced mast cell infiltration and intestinal damage. In the co-culture model, a substantial number of mast cells interact with the epithelial barrier, triggering activation of the IL-33/IL1RL1 (ST2) pathway and subsequent release of inflammatory factors and trypsin. This disruption leads to aberrant expression of tight junction proteins, which can be alleviated by supplementation with hucMSC-Ex. Our results suggest that hucMSC-Ex may reduce the release of mast cell mediators via the IL-33/IL1RL1 (ST2) axis, thereby mitigating its detrimental effects on intestinal barrier function.

Investigating the crucial role of selected Bifidobacterium probiotic strains in preventing or reducing inflammation by affecting the autophagy pathway.

Several studies have shown that probiotics can prevent and reduce inflammation in inflammation-related diseases. However, few studies have focused on the interaction between host and probiotics in modulating the immune system through autophagy. Therefore, we aimed to investigate the preventive and/or therapeutic effects of native potential probiotic breast milk-isolated Bifidobacterium spp. (i.e. B. bifidum, B. longum, and B. infantis) on the inflammatory cascade by affecting autophagy gene expression 24 and 48 h after treatment. Autophagy genes involved in different stages of the autophagy process were selected by quantitative polymerase chain reaction (qPCR). Gene expression investigation was accomplished by exposing the human colorectal adenocarcinoma cell line (HT-29) to sonicated pathogens (1.5×108 bacterial CFU ml-1) and adding Bifidobacterium spp. (MOI10) before, after, and simultaneously with induction of inflammation. An equal volume of RPMI medium was used as a control. Generally, our native potential probiotic Bifidobacterium spp. can increase the autophagy gene expression in comparison with pathogen. Moreover, an increase in gene expression was observed with our probiotic strains' consumption in all stages of autophagy. Totally, our selected Bifidobacterium spp. can increase autophagy gene expression before, simultaneously, and after the inflammation induction, so they can prevent and reduce inflammation in an in vitro model of inflammation.