HT-29 Cell Lines Research Articles

A Comparison of GAPDH and ACTB As Internal Control for Gene Expression Studies in Different Cancer Cell Lines

Housekeeping genes are used as internal controls to normalize the expression of target genes in gene expression studies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Actin beta (ACTB) are frequently preferred as housekeeping genes in gene expression studies. Due to the general alterations in the gene expression pattern in cancer cases, the selection of the appropriate housekeeping genes for these studies are challenging. In this study, we aimed to analyze the expression of the well-known housekeeping genes GAPDH and ACTB in 6 different cancer cell lines. Furthermore, the relative gene expression of the selected target gene (Tissue inhibitor of metalloproteinases 2-TIMP2) was normalized separately using GAPDH and ACTB and the obtained results were compared with each other. Finally, the stability of GAPDH and ACTB was analyzed using the in-silico tool, Bestkeeper. As a result of the study, it is found that the expression of GAPDH and ACTB were significantly different in the Jurkat (p< 0.01), MOLT4 (p< 0.05), REH (p< 0.001) and HT29 (p< 0.001) cell lines. Based on this finding, significantly different relative target gene expression values were obtained in different cell lines depending on whether the selected housekeeping gene was GAPDH or ACTB. In addition, GAPDH was found to show less variation among the samples used in all cell lines and more stability based on Bestkeeper analysis. These results support that the appropriate housekeeping gene selection, especially in cancer cell lines, may be an effective factor in obtaining accurate results for the studies in the field provide guidance to researchers. Keywords: Cancer cell lines, Gene expression, Housekeeping genes, GAPDH, ACTB

The JAK inhibitor, Tofacitinib, Corrects the Overexpression of CEACAM6 and Limits Susceptibility to AIEC Caused by Reduced Activity of the IBD Associated Gene, PTPN2.

A cohort of patients with inflammatory bowel disease (IBD) exhibit expansion of the gut pathobiont, adherent-invasive E. coli (AIEC). Loss of activity of the IBD susceptibility gene, protein tyrosine phosphatase type 2 ( PTPN2 ), results in dysbiosis of the gut microbiota both in human subjects and mice. Further, constitutive Ptpn2 knock-out ( Ptpn2 -KO) mice display expansion of AIEC compared to wildtype littermates. CEACAM6, a host cell surface glycoprotein, is exploited by AIEC to attach to and enter intestinal epithelial cells (IECs). Here, we investigate the role of IEC-specific PTPN2 in restricting AIEC invasion. Biopsies from IBD patients heterozygous (CT) or homozygous (CC) for the PTPN2 SNP (single nucleotide polymorphism) rs1893217 were processed for immunohistochemistry. HT-29 intestinal epithelial cells (IEC) were transfected with control shRNA ( PTPN2 -CTL), or a shRNA targeted towards PTPN2 ( PTPN2 -KD). The rs1893217 SNP was inserted ( PTPN2 -KI), or a complete knock-out of PTPN2 ( PTPN2 - KO) was generated, with CRISPR-Cas9 gene editing of Caco-2BBe IEC lines. Adherence and invasion assays were performed with either the human IBD AIEC isolate, LF82, or a novel fluorescent-tagged mouse adherent-invasive E. coli ( m AIEC red ) at multiplicity of infection (MOI) of 10. IL-6 and the pan-JAK inhibitor tofacitinib were administered to interrogate JAK-STAT signaling. Protein expression was determined by western blotting and densitometry. CEACAM6 expression was elevated (colon and ileum) in IBD patients carrying the PTPN2 rs1893217 SNP (CT, CC) compared to wildtype (TT) IBD patients. HT-29 and Caco-2BBe cell lines deficient in PTPN2 expressed significantly higher levels of CEACAM6. Further, PTPN2 -KI and PTPN2 -KO cell lines also displayed greater adherence and invasion by AIEC LF82 and higher m AIEC red invasion. CEACAM6 expression was further elevated after administration of IL-6 in PTPN2 -deficient cell lines compared to untreated controls. Silencing of STAT1 and 3 partially reduced CEACAM6 protein expression. Tofacitinib significantly reduced the elevated CEACAM6 protein expression and the higher AIEC adherence and invasion in PTPN2 -KI and PTPN2-KO cell lines compared to DMSO controls. Our findings highlight a crucial role for PTPN2 in restricting pathobiont entry into host cells. Our study also describes a role for the FDA-approved drug, tofacitinib (Xeljanz) in correcting the JAK-STAT- mediated over-expression of CEACAM6, used by pathobionts as an entry portal into host cells. These findings suggest a role for JAK-inhibitors in mitigating AIEC colonization in IBD-susceptible hosts.

Transcriptome Profiling Associated with CARD11 Overexpression in Colorectal Cancer Implicates a Potential Role for Tumor Immune Microenvironment and Cancer Pathways Modulation via NF-κB.

The immune system plays a critical role in inflammation by initiating responses to infections or tissue damage. The nuclear factor-κB (NF-κB) pathway plays a key role in inflammation and innate immunity, as well as other cellular activities. Dysregulation of this well-choreographed pathway has been implicated in various diseases, including cancer. CARD11 is a key molecule in the BCL10-MALT1 complex, which is involved in transducing the signal downstream of the NF-κB pathway. This study aims to elucidate how CARD11 overexpression exacerbates the prognosis of colorectal cancer (CRC). To identify the cellular pathways influenced by CARD11, transcriptomic analysis in both CRC cell lines and patients was carried out on CARD11- overexpressed HCT-116 and HT-29 CRC cell lines alongside empty vector-transfected cell lines. Furthermore, a comparison of transcriptomic data from adenoma and carcinoma CRC patients with low- (CARD11-) and high-(CARD11+) CARD11 expression was carried out. Whole transcriptomics and bioinformatics analysis results indicate that CARD11 appears to play a key role in CRC progression. Absolute GSEA (absGSEA) on HCT-116 transcriptomics data revealed that CARD11 overexpression promotes cell growth and tissue remodeling and enhances immune response. Key genes co-expressed with CARD11, such as EP300, KDM5A, HIF1A, NFKBIZ, and DUSP1, were identified as mediators of these processes. In the HT-29 cell line, CARD11 overexpression activated pathways involved in chemotaxis and extracellular matrix (ECM) organization, marked by IL1RN, MDK, SPP1, and chemokines like CXCL1, CXCL3, and CCL22, which were shown to contribute to the more invasive stage of CRC. In patient samples, adenoma patients exhibited increased expression of genes associated with the tumor immune microenvironment, such as IL6ST, collagen family members, and CRC transition markers, such as GLI3 and PIEZO2, in CARD11+ adenoma patients. Carcinoma patients showed a dramatic increase in the expression of MAPK8IP2 in CARD11+ carcinoma patients alongside other cancer-related genes, including EMB, EPHB6, and CPEB4.

Discovery and Prediction Study of the Dominant Pharmacological Action Organ of Aconitum carmichaeli Debeaux Using Multiple Bioinformatic Analyses.

Herbs, such as Aconitum carmichaeli Debeaux (ACD), have long been used as therapies, but it is difficult to identify which organs of the human body are affected by the various compounds. In this study, we predicted the organ where the drug predominantly acts using bioinformatics and verified it using transcriptomics. We constructed a computer-aided brain system network (BSN) and intestinal system network (ISN). We predicted the action points of ACD using network pharmacology (NP) analysis and predicted the dockable proteins acting in the BSN and ISN using statistical-based docking analysis. The predicted results were verified using ACD-induced transcriptome analysis. The predicted results showed that both the NP and docking analyses predominantly acted on the BSN and showed better hit rates in the hub nodes. In addition, we confirmed through verification experiments that the SW1783 cell line had more than 10 times more differentially expressed genes than the HT29 cell line and that the dominant acting organ is the brain, using network dimension spanning analysis. In conclusion, we found that ACD preferentially acts in the brain rather than in the intestine, and this multi-bioinformatics-based approach is expected to be used in future studies of drug efficacy and side effects.

Synthesis, cytotoxic evaluation, and in silico studies of novel benzenesulfonamide-thiazolidinone derivatives against colorectal carcinoma

Thirteen new benzenesulfonamide derivatives containing 4-thiazolidinone were synthesized. Their cytotoxic properties were tested on colorectal carcinoma (HT-29, DLD-1, COLO-205) and normal colon fibroblast (CCD-986Sk) cell lines. Compounds 3l (IC50=114.62 µM), 3a (IC50=25.82 µM), 3k (IC50=30.99 µM), and 3i (IC50=62.94 µM) showed the highest cytotoxicity on HT-29, DLD-1, COLO-205, and CCD-986Sk cell lines, respectively. Compound 3e (IC50=1075.4 µM) exhibited the least cytotoxicity against CCD-986Sk. The apoptosis profile of 5-FU at 5 µM was similar to that of compound 3e at 30 µM. Molecular docking and MD simulations showed stable interactions for compounds 3i and 3e. Molecular docking and MD simulations revealed that compounds 3i and 3e exhibit stable interactions with key cancer-related protein targets, particularly TGFβ2. These interactions suggest their potential as therapeutic agents. The predicted ADME parameters further highlighted compounds 3e and 3i for their favorable lipophilicity, solubility, permeability, and oral absorption profiles, supporting their promise as candidates for future drug development.

Phytochemical Study of Stachys iva Griseb. and In Vitro Evaluation of AhR Transcriptional Activity.

Genus Stachys L. consists of approximately 365 species, distributed mainly in temperate regions. Several members of this genus are widely used in the traditional medicine of different countries worldwide. In Greece, 54 Stachys taxa are found in parts of the mainland and/or insular country. The present study focused on the phytochemical investigation of Stachys iva Griseb. and the in vitro anti-inflammatory evaluation of the isolated compounds. In total, eighteen compounds were isolated and identified from the dichloromethane-methanol extract, belonging to iridoids, flavonoids, phenylethanoid glycosides, and phenolic acids. An in vitro approach assessed the aryl hydrocarbon receptor (AhR) modulatory effects of these compounds, revealing an AhR agonistic activity of the flavonoid aglycones apigenin and cirsimaritin in HepG2 and HT29 cell lines. The present study contributes to the evidence of the traditional uses of Stachys spp. and its bioactive constituents, justifying the ethnopharmacological use as an anti-inflammatory plant genus.

Antioxidant and Biological Activity of Mexican Madroño Fruit (Arbutus arizonica).

The fruit of the Mexican madroño (Arbutus arizonica) has been consumed since pre-Columbian times by North American tribes and native groups in Mexico. Despite this, reports on its chemical composition and biological activity are limited. This work aims to determine the antioxidant, antiproliferative, and digestive enzyme inhibition activities of the methanol amberlite-retained extract of Mexican madroño. Results showed that madroño fruit is rich in antioxidants: DPPH (EC50 = 0.89 ± 0.03 mg/mL), TEAC (1078 ± 4.9 μM/g), and hemolysis inhibition (IC50 = 358.07 μg/mL), with high phenolic and flavonoid content at 15.92 ± 3.2 mg GAE/g and 4.33 ± 0.3 mg CA/g, respectively. Using analytical chromatography, gallic acid, vanillic acid, chlorogenic acid, ferulic acid, quercetin, and rutin were quantified. The extract also showed α-glucosidase inhibition (IC50 = 3.1 ± 0.17 mg/mL), but no inhibition against α-amylase and lipase (>5 mg/mL), while showing antiproliferative activity against HeLa, HT-29, and MCF-7 cancer cell lines. These results point towards an interesting potential for the fruit of the A. arizonica as chemopreventive and hold potential for elaborating functional foods.

Synthesis, Characterization, and Biological Evaluation of Novel [M(η6-arene)2]+ (M = Re, 99mTc) Hosted Terpyridines and Copper Complexes Thereof.

We report the synthesis, characterization, and in vitro biological activities of [Re(η6-arene)2]+-terpyridine conjugates and their CuII complexes. The terpyridine (terpy) chelators were attached to the [Re(η6-arene)2]+ scaffold via secondary amine linkers allowing for heteroleptic mono- and homoleptic bis-terpyridine-substituted chelators. Complexation with CuCl2 afforded the respective square pyramidal [Cu(terpy)Cl2] complexes hosted on the [Re(η6-arene)2]+ scaffold. The chelator conjugates and their respective complexes were found to be remarkably cytotoxic against malignant HT29 and A549 human cancer cell lines in vitro with IC50 values in the low micromolar range. Mitochondrial respiration disruption was identified as a possible mode of action of these novel drug candidates. Crucially, the [Re(η6-arene)2]+ hosts delivered water solubility of the otherwise insoluble [Cu(terpy)Cl2] motif. Importantly, the homoleptic [99mTc(η6-arene)2]+-terpyridine conjugate is available in a single step, which enables the presented system to be used as a theranostic approach to modern medicinal inorganic chemistry.

Design, Synthesis and In-Vitro Antimicrobial and Cytotoxic Activity Screening of 5-Carboxamide Substituted 3, 4-Dihydropyrimidine-2 (1H) Ones

This paper describes the synthesis and in vitro biological evaluation of new compounds designated as 5(a-h) and 8(a-f). These compounds were synthesized by the reaction of thiazole derivative (3) or thiadiazole derivative (7) with urea and various aromatic aldehydes 4(a-h) in ethanol. The structures of the synthesized compounds were checked by IR,1H,13C NMR, and Mass spectroscopy. The antibacterial and antifungal activities of the compounds were evaluated against Escherichia coli (Gram-negative), Staphylococcus aureus (Gram-positive), and Candida albicans (fungus) using agar well diffusion, minimum inhibitory concentration (MIC), and minimum bactericidal/fungicidal concentration methods. The cytotoxic activities of these compounds against HT-29 and A-549 cancer cell lines were assessed in vitro by the MTT method. Our studies showed compounds 8c and 8f to have the most expressed antibacterial activity against S. aureus, while compounds 8c and 8d were the most potent against E. coli. Compounds 5h and 8e showed the highest antifungal activity against C. albicans, and compound 5f showed the most potent activity against the tested cancer cell lines.

Cytotoxic Activity and Phytochemical Analysis of Artemisia biennis Essential Oils at Different Growth Stages

Background: The Artemisia genus is well-known for its medicinal properties, particularly in Iranian traditional medicine. Artemisia biennis, a species within this genus, is widely distributed across Iranian rangelands. Objectives: This study aimed to evaluate the cytotoxic activity and chemical composition of essential oils (EO) isolated from the aerial parts of A. biennis in Iran at different growth stages. Methods: The aerial parts of A. biennis from northeast Iran were collected during June (early vegetative stage), July (pre-flowering stage), August (full-flowering stage), and October (late vegetative stage). The essential oils (EOs) of the A. biennis species were extracted by hydro-distillation using a Clevenger-type apparatus and analyzed using gas chromatography–mass spectrometry (GC/MS). The cytotoxic activity of the EOs against normal fibroblasts, MCF-7, and HT-29 cell lines was evaluated using the MTT assay. Results: Data from GC/MS analysis revealed that (Z)-nerolidol (22.62 - 54.4%), (E)-β-farnesene (6.89-16.38%), and (Z)-tonghaosu (12.33 - 18.61%) were the most abundant chemical constituents. The essential oil (EO) of A. biennis was characterized by a high content of oxygenated sesquiterpenes, with (Z)-nerolidol being the main constituent identified. Based on the MTT assay, the EO of the plant species collected in June exhibited the most potent cytotoxicity against MCF-7 (IC50 = 2.84 ± 0.15 µg/mL) and HT-29 cell lines (IC50 = 2.41 ± 0.2 µg/mL). Conclusions: The growth stage of A. biennis affects EO yields, the composition of extracted secondary metabolites, and cytotoxic activity. A. biennis EOs can be considered potential sources of cytotoxic phytochemicals.

Investigating the anticancer potential and apoptotic signaling of nanocapsule-loaded pyrogallol in ovarian cancer cells (A2780)

Nanoencapsulation technology enables the efficient delivery of natural bioactive compounds, enhancing stability and controlled release to improve bioavailability and therapeutic efficacy. Pyrogallol, a natural polyphenolic compound, has shown promising anticancer activities against various cancer cell lines. The purpose of this study was to synthesize and characterize nanocapsule-encapsulated pyrogallol (Na-Pyr) and investigate its anticancer potential, apoptotic signaling, and anti-metastatic properties. The process involved synthesizing the nanocapsules using the sol–gel method and evaluating their physical and chemical properties using various techniques. The MTT assay was used to test Na-Pyr’s cytotoxicity against various cancer cell lines, including ovarian cancer (A2780), human colorectal adenocarcinoma (HT-29), and pancreatic cancer (ASPC-1), with Huvec serving as the normal cell line. DAPI staining, real-time PCR, and flow cytometry were performed to examine apoptosis induction and cell cycle arrest. The results obtained from dynamic light scattering (DLS) manifested that Na-Pyr had a size of 176.4 ± 7.26 nm, an average zeta potential of about 42.1 ± 2.01 mV, and a polydispersity (PDI) index of 0.174 ± 0.02. Electron microscopy images exhibited a smooth surface and spherical shape, indicating material compatibility. Na-Pyr exhibited higher anticancer potential against ovarian cancer cells than HT-29 and ASPC-1 cancer cell lines with an IC50 value of 23.5 ± 0.95 μg/mL. Na-Pyr significantly modulates the Bax/Bcl2 ratio and activates the MMP9 pathway-mediated apoptosis. Flow cytometry analysis supported these findings, revealing increased percentages of necrotic, early apoptotic, and late apoptotic populations upon exposure to Na-Pyr. In conclusion, the study highlights the promising anticancer potential of Na-Pyr and its potential as an effective treatment strategy.

The NRF2-KEAP1 pathway and epigenetic control in colorectal cancer: A promising prognostic and predictive biomarker

In colorectal cancer (CRC), important genes in the NRF2/KEAP1 pathway may undergo methylation, yet the exact genes that control this pathway in CRC are not entirely understood. Targeting the NRF2/KEAP1 pathway could be a valuable therapeutic strategy for colorectal cancer. Our research examined the methylation and expression of the KEAP1 gene in precancerous lesions and colorectal cancer, as well as investigated this pathway in the HT-29 cell line for potential CRC treatment. In this study, 208 frozen colorectal tissue samples were analysed, including 34 tumours, 60 precancerous lesions with adjacent normal tissues, and 20 normal tissue samples. The methylation-specific PCR and quantitative PCR were used to examine KEAP1 gene promoter methylation, and NRF2 and KEAP1 gene expressions, respectively. Furthermore, the HT 29 cell line was treated with 5-Azacytidine, followed by quantitative real-time PCR to evaluate post-demethylation KEAP1 and NRF2 mRNA expression. Unlike NRF2, our research revealed a notable reduction in KEAP1 gene expression in CRC tissues compared to adjacent normal tissues (p=0.02). Intriguingly, treating the HT-29 cell line with 5-Azacytidine led to a significant increase in KEAP1 gene expression compared to untreated cells (P=0.03), with no significant alteration observed in NRF2 gene expression. Examination of CpG islands within the KEAP1 gene promoter indicated extensive hypermethylation in 58.8% of tumour tissues compared to adjacent normal samples. These findings indicate that decreased KEAP1 gene expression resulting from promoter hypermethylation could interfere with the NRF2/KEAP1 pathway, a crucial mechanism in colorectal carcinogenesis. The study proposes that KEAP1 may play a significant role in the progression of CRC and could potentially serve as a biomarker for diagnosing and monitoring therapy in CRC

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.

Development of a novel oncolytic adenovirus controlled by CDX2 promoter for esophageal adenocarcinoma therapy.

Prognosis of esophageal adenocarcinoma (EAC) is still poor. Therefore, the development of novel therapeutic modalities is necessary to improve therapeutic outcomes in EAC. Here, we report a novel promoter-controlled oncolytic adenovirus targeting CDX2 (Ad5/3-pCDX2) and its specific anticancer effect for EAC. We used OE19, OE33, HT29, MKN28, RH30, and HEL299 cell lines. To establish CDX2 overexpressing OE19 cells, pCMV-GLI1 plasmid was transfected to OE19 (OE19 + GLI1). The virus replication and cytocidal effect of replication competent Ad5/3-pCDX2 were analyzed in vitro. Antitumor effect of Ad5/3-pCDX2 was assessed in xenograft mouse models by intratumoral injection of the viruses. Finally, efficacy of combination therapy with Ad5/3-pCDX2 and 5FU was evaluated. EAC cells and HT29 showed high mRNA levels of CDX2, but not MKN28, RH30, and HEL299. We confirmed that deoxycholic acid (DCA) exposure enhanced CDX2 expression in EAC cells and OE19 + GLI1 had persistent CDX2 overexpression without DCA. Ad5/3-pCDX2 showed stronger cytocidal effect in OE19 + GLI1 than OE19, whereas Ad5/3-pCDX2 did not kill CDX2-negative cells. Ad5/3-pCDX2 was significantly replicated in EAC cells and the virus replication was higher in OE19 + GLI1 and OE19 with DCA compared to OE19 without DCA exposure. In vivo, Ad5/3-pCDX2 significantly suppressed OE19 tumor growth and the antitumor effect was enhanced in OE19 + GLI1 tumor. In contrast, Ad5/3-pCDX2 did not show significant antitumor effect in MKN28 tumor. Moreover, Ad5/3-pCDX2 significantly increased the efficacy of 5FU in vitro and in vivo. Ad5/3-pCDX2 showed specific anticancer effect for EAC, which was enhanced by bile acid exposure. Ad5/3-pCDX2 has promising potential for EAC therapy in the clinical setting.

Comparative analysis of bisphenol A effects on HT-29 cell line and mouse intestinal organoids

Comparative analysis of bisphenol A effects on HT-29 cell line and mouse intestinal organoids

Short-chain fructooligosaccharides synthesis using a commercial enzyme complex from Aspergillus sp. and anti-cancer activity on HCT116 and HT-29 cell lines

Over the last years, short-chain fructooligosaccharides (FOS) have gained significant attention as valuable food and dietary supplement components. Apart from implications such as dietary fibres, sweeteners, and humectants, FOS are hailed for their prebiotic properties. Moreover, much research is dedicated to their role as anti-cancer agents. Despite the ongoing interest, both technical aspects, challenges of FOS production, and their anti-cancer role necessitate more in-depth investigations to understand their healthcare role better. This study aimed to optimise critical FOS synthesis parameters using a commercial enzyme, Viscozyme® L, to obtain a preparation with a high FOS content. A central composite design and response surface methodology evaluated the effect of pH, temperature, synthesis time, substrate, and enzyme load on multiple responses, chosen as independent variables, were utilised to define optimal conditions. Under the optimal conditions (temperature 50 °C, pH 5.5, sucrose concentration 352 g/L, enzyme concentration 2.5% v/v, and 5.5 h of reaction), a preparation with a total FOS content of 58.8% was obtained. The FOS preparation showed minimal in vitro antioxidant capacity in the ORAC, CUPRAC, and ABTS*+assays. In addition, the cytotoxicity of FOS against tested cell lines was evaluated. FOS did not reduce HT-29 cell viability even at the concentration of 2.5 mg/mL, whereas FOS inhibited the proliferation of HCT116 cells by 50% at 2.35 mg/mL. Finally, the sensitisation effect of FOS on HCT116 cell treatment with doxorubicin has been revealed.

Oncofetal morphogenesis similar to embryonic gut formation by a subpopulation of DLD-1 human colon cancer cells

Cancer stem cells (CSC) are thought to be responsible for cancer phenotypes and cellular heterogeneity. Here we demonstrate that the human colon cancer cell line DLD1 contains two types of CSC-like cells that undergo distinct morphogenesis in the reconstituted basement membrane gel Matrigel. In our method with cancer cell spheroids, the parent cell line (DLD1-P) developed grape-like budding structures, whereas the other (DLD1-Wm) and its single-cell clones dynamically developed worm-like ones. Gene expression analysis suggested that the former mimicked intestinal crypt-villus morphogenesis, while the latter mimicked embryonic hindgut development. The organoids of DLD1-Wm cells rapidly extended in two opposite directions by expressing dipolar proteolytic activity. The invasive morphogenesis required the expression of MMP-2 and CD133 genes and ROCK activity. These cells also exhibited gastrula-like morphogenesis even in two-dimensional cultures without Matrigel. Moreover, the two DLD1 cell lines showed clear differences in cellular growth, tumor growth and susceptibility to paclitaxel. This study also provides a simple organoid culture method for human cancer cell lines. HT-29 and other cancer cell lines underwent characteristic morphogenesis in direct contact with normal fibroblasts. Such organoid cultures would be useful for investigating the nature of CSCs and for screening anti-cancer drugs. Our results lead to the hypothesis that CSC-like cells with both invasive activity and a fetal phenotype, i. e. oncofetal CSCs, are generated in some types of colon cancers.

Isothiocyanates attenuate heparin-induced proliferation of colon cancer cells invitro.

Isothiocyanates (ITCs), prevalent in cruciferous vegetables, are known for their anticarcinogenic properties. Prior research has indicated that heparin can stimulate the growth of colon cancer cells. However, the implications of ITCs in the diet of cancer patients receiving heparin-based therapies have yet to be fully understood. This exploratory invitro study examines the proliferative effects of low-molecular-weight heparin (LMWH) on human colon cancer cells and assesses the antiproliferative potential of four ITC compounds, exploring possible epidermal growth factor family of receptor tyrosine kinases (Erb-B) related mechanisms. We evaluated cell viability in HCT-116 and HT-29 cell lines following treatment with ITCs alone or combined with LMWH (20 μg/mL) at various concentrations (1-100 μM). Clonogenic and wound-healing assays were performed after 24 h of treatment with 5 μM ITCs. Additionally, messenger RNA (mRNA) and protein expression of Erb-B family genes was measured using quantitative polymerase chain reaction (qPCR) and Western blotting. Statistical analysis was conducted using analysis of variance (ANOVA) with Dunnett's post hoc test. Results indicated that the half-maximal inhibitory concentration (IC50) values for Phenylethyl isothiocyanate (PEITC), Benzyl isothiocyanate (BITC), and Sulforaphane (SFN) were lower than those of Allyl isothiocyanate (AITC) in LMWH-stimulated HCT-116 (20.77, 19.10, and 44.05 μM, respectively) and HT-29 (74.94, 26.77, and 43.49 μM, respectively). PEITC and SFN significantly reduced ErbB1 (epidermal growth factor receptor (EGFR)) and ErbB4 (receptor tyrosine-protein kinase erbB-4) expression, while BITC decreased ErbB2 (receptor tyrosine-protein kinase erbB-2) and transforming growth factor beta (TGF-β) expression in HCT-116 cells (all, p < .05). PEITC, BITC, and SFN also increased proapoptotic Bax expression and decreased the antiapoptotic B-cell lymphoma 2 (Bcl-2) expression (all, p < .05). These findings suggest that specific ITCs may mitigate cancer cell proliferation induced by LMWH in cancer therapies, highlighting their potential therapeutic efficacy.

A Promising Approach to Target Colorectal Cancer Using Hybrid Triarylmethanes.

Aiming to create an innovative series of anti-colorectal cancer agents, we designed in this work hybrid triarylmethane compounds. Three hybrid triarylmethanes and their corresponding N-oxide analogues were successfully synthesized using an efficient procedure that involved connecting two triarylmethane molecules, through mono-, bi-, and triethylene glycol fragments. In our pursuit to develop more soluble molecules, we synthesized a hybrid triarylmethane featuring a lysine-based spacer through a convergent strategy involving 7 steps. All hybrid compounds were assessed for their antiproliferative activity on human HT-29 and HCT116 colorectal cancer (CRC) cell lines. Three pyridine N-oxide analogs demonstrated notable antiproliferative potential among the set of tested compounds, with IC50 values ranging from 18 to 24 μM on both human CRC cell lines analyzed. A cytotoxicity study conducted on murine fibroblasts revealed that these three active compounds were not toxic at the IC50 values, indicating their suitability for further drug development. A docking study was conducted on two representative compounds, one for each series and protein kinase B (AKT) was identified as a potential target of their in anti-cancer effects. A computational drug-likeness study predicted favourable oral and intestinal absorption efficiency.

Efficacy assessment of a novel pan-RAS inhibitor in KRAS-mutant and wild type colorectal 3D bioprinted organoid tumor tissue.

91 Background: Colorectal cancer (CRC) is the third leading type of cancer worldwide, with ~150,000 new cases in the US annually and a grim 14% 5-year survival for patients diagnosed at a late stage. A lack of treatment options leads to persistently poor prognosis for patients with advanced stage disease. KRAS mutations are well known drivers of CRC and other GI cancers. Multiple KRAS mutations occur in CRC, including G12D (34%), G12V (21%), G13D (20%), G12C (8%), and others (18%). Existing KRAS-targeted therapies have limited use in CRC, underscoring the need for pan-RAS inhibitors in treating CRC and other RAS driven cancers. Objective: Assess activity of ADT-007, our pan-RAS inhibitor, on wild-type (WT) and KRAS-mutant 3D bioprinted organoid tumor (BOT) tissue using our high-throughput ex vivo platform. Methods: Using previously established bioprinting protocols, WT and mutant BOTs were printed with HT29 and HCT116 cells, respectively. HT29 is an established human WT CRC cell line with known sensitivity to proteosome and survivin inhibitors. HCT116 is a KRASG13D mutant human CRC cell line. 3 sets of BOTs were generated and acclimated for 24h. One set was treated for 72h with proteosome inhibitor Bortezomib, another with survivin inhibitor YM155, and the third with our novel pan-RAS inhibitor ADT-007. Dose response curves were generated from both conventional ATP luminescence readouts and high-content imaging. Results: BOT tissue microarchitecture was validated and &gt;200 µm diffusion in BOTs was confirmed using high-content imaging. Differential response was quantified using Cell TiterGlo endpoint assay as well as advanced image processing of high-content live/dead nuclear stained images captured at multiple z-plains. ADT-007 IC50 was found to be substantially lower for mutant HCT116 compared to that for WT HT29 cell line BOTs, which was consistent with separately conducted in vitro and in vivo studies. Conclusions: A pan-RAS inhibitor, such as ADT-007 with high selectivity for cancer cells with activated RAS that is not limited to a specific KRAS mutant allele or RAS isozyme, could have broader use for CRC and other RAS-driven cancers. Further, due to their potential to replicate biophysical characteristics of a tumor and its microenvironment, BOT based precision and personalized medicine platforms can provide more accurate drug efficacy readout compared to in vitro cancer models.