Colon Adenocarcinoma Cells Research Articles

Optimizing the Design and Geometry of T Cell-Engaging Bispecific Antibodies Targeting CEA in Colorectal Cancer.

Metastatic colorectal cancer remains a leading cause of cancer-related deaths, with a 5-year survival rate of only 15%. T cell-engaging bispecific antibodies (TCBs) represent a class of biopharmaceuticals that redirect cytotoxic T cells toward tumor cells, thereby turning immunologically "cold" tumors into "hot" ones. The carcinoembryonic antigen (CEA) is an attractive tumor-associated antigen that is overexpressed in more than 98% of patients with colorectal cancer. In this study, we report the comparison of four different TCB formats employing the antibodies F4 (targeting human CEA) and 2C11 (targeting mouse CD3ε). These formats include both antibody fragment-based and IgG-based constructs, with either one or two binding specificities of the respective antibodies. The 2 + 1 arrangement, using an anti-CEA single-chain diabody fused to an anti-CD3 single-chain variable fragment, emerged as the most potent design, showing tumor killing at subnanomolar concentrations across three different CEA+ cell lines. The in vitro activity was three times greater in C57BL/6 mouse colon adenocarcinoma cells (MC38) expressing high levels of CEA compared with those expressing low levels, highlighting the impact of CEA density in this assay. The optimal TCB candidate was tested in two different immunocompetent mouse models of colorectal cancer and showed tumor growth retardation. Ex vivo analysis of tumor infiltrates showed an increase in CD4+ and CD8+ T cells upon TCB treatment. This study suggests that bivalent tumor targeting, monovalent T-cell targeting, and a short spatial separation are promising characteristics for CEA-targeting TCBs.

Effects of Reduced Extracellular Sodium Concentrations on Cisplatin Treatment in Human Tumor Cells: The Role of Autophagy.

Hyponatremia is the prevalent electrolyte imbalance in cancer patients, and it is associated with a worse outcome. Notably, emerging clinical evidence suggests that hyponatremia adversely influences the response to anticancer treatments. Therefore, this study aims to investigate how reduced extracellular [Na+] affects the responsiveness of different cancer cell lines (from human colon adenocarcinoma, neuroblastoma, and small cell lung cancer) to cisplatin and the underlying potential mechanisms. Cisplatin dose-response curves revealed higher IC50 in low [Na+] than normal [Na+]. Accordingly, cisplatin treatment was less effective in counteracting the proliferation and migration of tumor cells when cultured in low [Na+], as demonstrated by colony formation and invasion assays. In addition, the expression analysis of proteins involved in autophagosome-lysosome formation and the visualization of lysosomal areas by electron microscopy revealed that one of the main mechanisms involved in chemoresistance to cisplatin is the promotion of autophagy. In conclusion, our data first demonstrate that the antitumoral effect of cisplatin is markedly reduced in low [Na+] and that autophagy is an important mechanism of drug escape. This study indicates the role of hyponatremia in cisplatin chemoresistance and reinforces the recommendation to correct this electrolyte alteration in cancer patients.

Genotoxic and antiproliferative properties of Endopleura uchi bark aqueous extract

ABSTRACT The bark extract from Endopleura uchi has been widely used in traditional medicine to treat gynecological-related disorders, diabetes, and dyslipidemias albeit without scientific proof. In addition, E. uchi bark extract safety, especially regarding mutagenic activities, is not known. The aim of this study was to determine the chemical composition, antitumor, and toxicological parameters attributed to an E. uchi bark aqueous extract. The phytochemical constitution was assessed by colorimetric and chromatographic analyzes. The antiproliferative effect was determined using sulforhodamine B (SRB) assay using 4 cancer cell lines. Cytotoxic and genotoxic activities were assessed utilizing MTT and comet assays, respectively, while mutagenicity was determined through micronucleus and Salmonella/microsome assays. The chromatographic analysis detected predominantly the presence of gallic acid and isoquercitrin. The antiproliferative effect was more pronounced in human colon adenocarcinoma (HT-29) and human breast cancer (MCF-7) cell lines. In the MTT assay, the extract presented an IC50 = 39.1 µg/ml and exhibited genotoxic (comet assay) and mutagenic (micronucleus test) activities at 20 and 40 µg/ml in mouse fibroblast cell line (L929) and mutagenicity in the TA102 and TA97a strains in the absence of S9 mix. Data demonstrated that E. uchi bark possesses bioactive compounds which exert cytotoxic and genotoxic effects that might be associated with its antitumor potential. Therefore, E. uchi bark aqueous extract consumption needs to be approached with caution in therapeutic applications.

Antitumoral activity of different Amaryllidaceae alkaloids: In vitro and in silico assays

Ethnopharmacology relevanceThe plants of Amaryllidaceae family, such as Amaryllis belladonna L., have been used as herbal remedies for thousands of years to address various disorders, including diseases that might today be identified as cancer. Aim of the studyThe objective of this work was to evaluate the potential of three Amaryllidaceae alkaloids against four cancer cell lines. Material and methodsThe alkaloids lycorine, 1-O-acetylcaranine, and montanine were evaluated in vitro against colon adenocarcinoma cell line (HCT-116) and breast carcinoma cell lines (MCF-7, MDAMB231, and Hs578T). Computational experiments (target prediction and molecular docking) were conducted to gain a deeper comprehension of possible interactions between these alkaloids and potential targets associated with these tumor cells. ResultsMontanine presented the best results against HCT-116, MDAMB231, and Hs578T cell lines, while lycorine was the most active against MCF-7. In alignment with the target prediction outcomes and existing literature, four potential targets were chosen for the molecular docking analysis: CDK8, EGFR, ER-alpha, and dCK. The docking scores revealed two potential targets for the alkaloids with scores similar to co-crystallized inhibitors and substrates: CDK8 and dCK. A visual analysis of the optimal docked configurations indicates that the alkaloids may interact with some key residues in contrast to the other docked compounds. This observation implies their potential to bind effectively to both targets. ConclusionsIn vitro and in silico results corroborate with data literature suggesting the Amaryllidaceae alkaloids as interesting molecules with antitumoral properties, especially montanine, which showed the best in vitro results against colorectal and breast carcinoma. More studies are necessary to confirm the targets and pharmaceutical potential of montanine against these cancer cell lines.

Cytotoxicity Analysis of the Effects of Heterobasidion Annosum Mycelia and Cisplatin on Colon Adenocarcinoma (CACO-2) Cell Line

Colorectal cancer ranks as the third most prevalent form of cancer and stands as the second leading cause of mortality. Both environmental and genetic risk factors contribute to its manifestation. Presently, 5-fluoruracil/leucovorin (5-FU/LV) remains the recommended course for adjuvant therapy in addressing this condition. Conversely, mushrooms, celebrated for their biologically active constituents, including valuable enzymes, have emerged as a captivating subject in diverse medical disciplines, particularly within the realm of cancer therapy, due to their promising therapeutic properties. This specific investigation aimed to conduct in vitro cytotoxic experiments using extracts obtained from Heterobasidion annosum micelles cultivated in a liquid malt extract medium. The pulverized extracts were dissolved in Dulbecco's Modified Eagle Medium (DMEM) at varied concentrations ranging from 25ng/mL to 200ng/mL and subsequently administered to colon adenocarcinoma (Caco-2) cells. The cytotoxic effects of both the fungus and cisplatin, a well-known anticarcinogenic agent, were examined at intervals of 24, 48, and 72 hours. The findings indicated a significant inhibition of cancer cell development within this timeframe. Moreover, a noteworthy discovery emerged, revealing that cisplatin, known for its efficacy in various cancer studies, substantially diminished the viability of cancer cells after 72 hours in comparison to the control group.

Abstract 7402: Foundation AI models predict molecular measurements of tumor purity

Abstract Background: Molecular assays, such as comprehensive genomic profiling, play a critical role in characterizing patient disease and guiding treatment selection in oncology but require sufficient tumor nucleic acids in tissue samples for reliable results. Automated, accurate and reproducible quantification of tissue area and tumor nuclei from H&E slides to determine suitability for molecular testing could reduce the amount of time spent assessing tissue quality. To address this need, we developed pan-indication foundation AI models to quantify tissue and cell features within H&E whole-slide images (WSI) and compared tumor purity estimates to orthogonal molecular and manual methods. Methods: Pan-indication artifact, tissue, and cell models were each trained using at least 2.8 million pathologist annotations across over 100,000 WSI from 14 stains, 25 organs, and 9 scanners and validated against pathologist ground-truth labels. This suite of foundation models was deployed on H&E WSIs of colon adenocarcinoma (COAD; N=442), melanoma (N=363), prostate cancer (PRAD; N=490), and non-small cell lung cancer (NSCLC; N=996) from TCGA1. Model outputs allowed for the segmentation of tumor regions and enumeration of cancer cells for the purpose of determining tumor purity scores, calculated as the fraction of cancer cells within tumor tissue on a WSI. For comparison, tumor purity values determined based on DNA copy number (ABSOLUTE), gene expression (ESTIMATE), DNA methylation (LUMP) and manual assessment2 were obtained. Concordance between AI model scores and other tumor purity values was assessed using the intraclass correlation coefficient, ICC(2,1). Results: AI model scores were correlated with orthogonal molecular measures of tumor purity in all four indications. AI model purity scores corresponded the most with ABSOLUTE (ICC range: 0.22-0.46), followed by LUMP (ICC range: 0.02-0.50) and ESTIMATE (ICC range: 0.03-0.34). Across indications, the highest agreement observed between AI model scores and molecular methods was in melanoma (ICC range: 0.34-0.50), while the lowest agreement was in PRAD (ICC range: 0.02-0.22). Compared against ABSOLUTE in NSCLC and PRAD, AI model scores showed the highest concordance, while the other two molecular methods and manual assessment all overestimated ABSOLUTE tumor purity. Conclusions: Here, we describe a pan-indication AI-based approach for the quantification of tumor features in H&E-stained WSI. Foundation AI models successfully segment tumor tissue regions and enumerate cancer cells for determining tumor purity, and these predictions correlate with molecular metrics in four cancer indications. This work provides further evidence of how AI could improve the efficiency of molecular testing and enhance precision medicine approaches in oncology.

Abstract 7126: Synergistic combinations and tissue type-specificity of compounds targeting components of the DNA damage response pathway in 2D culture and 3D spheroid cell viability assays

Abstract Poly (ADP-ribose) polymerase (PARP) inhibitors have changed the landscape of treatment for homologous recombination (HR)-deficient cancers. PARP inhibitors are synthetically lethal with deficiencies in genes involved in the HR pathway, such as BRCA2, but resistance against PARP inhibitors is a major problem in the clinic. The use of new synergistic drug combinations targeting different components of the DNA damage response (DDR) pathway may overcome PARP inhibitor resistance. Sixteen small molecule inhibitors acting on different targets in the DDR pathway (including PARP, Polθ and ATR) were profiled in 2D culture and 3D spheroid cell viability assays. Intracellular ATP content was used as an indirect readout of cell number. Compound synergies were determined by profiling combinations of compounds in a 6-by-6 matrix, and quantifying synergistic effects using the Excess over Bliss score [1]. A pair of isogenic BRCA2-deficient and -proficient DLD-1 colon adenocarcinoma cell lines was used as a model of BRCA2-deficient tumors. In 2D cell viability assays, PARP inhibitors and the Polθ inhibitor ART558 were more active in BRCA2-deficient compared to BRCA2-proficient DLD-1 cells, whereas an ATR inhibitor (ceralasertib) was equally potent in both cell lines. Combining olaparib with ART558 in 2D culture and 3D spheroid studies showed synergistic effects in BRCA2-deficient DLD-1 cells. Moreover, olaparib synergized with ceralasertib in both BRCA2-deficient and -proficient DLD-1 cells. Different DDR-targeting inhibitors were profiled in over a hundred cancer cell lines from diverse tumor tissue origin in 2D assays. This showed a relatively high potency for PARP inhibitors in ovarian cancer cell lines compared to cell lines derived from other tissue types, which is consistent with the clinical application of PARP inhibitors for treatment of ovarian cancers. For ATR inhibitors and ART558, kidney and myeloid cancer cell lines were found to be most sensitive, respectively, suggesting potential therapeutic utility of ATR and Polθ inhibitors for treatment of these cancer types. The genomic determinants of the different sensitivities were investigated by comparing compound potencies in the cell viability assays with the mutation status of more than 500 genes involved in cancer or the DDR pathway in the cell lines. Additionally, cancer cell line sensitivity was correlated with the expression levels of more than 19,000 genes in these cell lines to identify novel RNA signatures capable of predicting response to DDR-targeting inhibitors. This study uncovers new synergistic drug combinations that increase the efficiency of PARP inhibitors, and provides new insights into the determinants of cancer cell line sensitivity to compounds targeting different components of the DDR pathway. [1] Liu et al. Stat. Biopharm. Res. 10, 112-22; 2018 Citation Format: Janneke J. Melis, Daphne J. Kluitmans, Jeffrey J. Kooijman, Jelle Dylus, Sabine Wetemans, Suzan Geerdink-Datema, Jeroen A. de Roos, Yvonne Grobben, Guido J. Zaman. Synergistic combinations and tissue type-specificity of compounds targeting components of the DNA damage response pathway in 2D culture and 3D spheroid cell viability assays [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 7126.

Abstract 2170: Effects of folic acid supplementation on early stages of colitis-associated carcinogenesis

Abstract Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) characterized by persistent mucosal inflammation extending throughout the colon. Due to compromised intestinal surface integrity and the prolonged use of medication, individuals with UC often develop a folate deficiency, requiring folic acid (FA) supplementation. Prior findings from this group indicate that FA supplementation, at clinically relevant levels, promotes UC-associated dysplasia. Supplementation with FA resulted in an increased multiplicity of dysplasia and a significant elevation in the expression of several inflammatory genes (e.g. IL-6, Cox-2, Iκκ-β) within the colonic mucosa of mice with UC. Genome-wide expression profiling of normal and dysplastic colonic epithelial cells from mice treated with different doses of FA revealed differentially expressed genes were enriched for members of the ERK and NF-κB pathways. Subsequent in vitro analyses, utilizing isogenic RKO colon adenocarcinoma cells exposed to various doses of FA, demonstrated the downregulation of ERK and p-ERK in wild type (WT) p53+/+ cells, while an increase in expression was observed in p53−/− cells at the protein level. The goal of this study is to investigate the role of the MAPK/ERK pathway in the development of UC-associated neoplasms. An in vitro model that faithfully reproduces the inflamed microenvironment in the context of WT vs. mutant p53, the putative gatekeeper of UC-associated carcinogenesis, is needed to enhance our understanding of early changes in the colon that are induced by FA and drive tumor formation. Colon organoids are being generated by isolating crypts from WT p53+/+ and mutant p53+/515A mice. These organoids are then co-cultured with mouse macrophages (RAW264.7 cells) in inserts to stimulate crosstalk between the macrophages and colonic epithelial cells, as in the inflamed colon. Analyses demonstrate that the macrophages express pivotal inflammatory cytokines characteristic of UC, including IL-1β, IL-10, TNF-α, and IL-6, irrespective of the presence or absence of LPS stimulation. Co-cultured organoids and macrophages will be exposed to different concentrations of FA for various lengths of time, and the impact of treatment on the levels of ERK, p-ERK, and p65 will be assessed. The ability of p-ERK to mediate the transcriptional activation of NF-κB and its downstream effector IL-6 will also be evaluated. Subsequent investigations employing this innovative experimental model will reveal the mechanisms by which FA supplementation promotes the progression of UC-associated dysplasia. Given the potential risks associated with FA supplementation in individuals with UC, there is an urgent need to translate these results to a clinical setting. (Supported by CA262551 and the Timothy and Aurora Hughes Cancer Research Fund). Citation Format: Ariane Rocha Bartolomeu, Wen-Chi Chang, Lisa Vanderveer, Kristen N. Harvey, Mitchell Cheung, Harry S. Cooper, Margie Lee Clapper. Effects of folic acid supplementation on early stages of colitis-associated carcinogenesis [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 2170.

Abstract 3175: Dear ABBIE, edit that colon cancer

Abstract Genome editing has exciting potential for gene therapy of cancers. Colorectal cancer is the third most occurring cancer in both men and women. The current treatments for colon cancers include surgery, radiofrequency ablation, chemotherapies and immunotherapies. These either involve removal of intestinal tissue that can alter the quality of life of the patient, or require doses of drugs that generally result in various side effects. ABBIE (A Binding Based Integrase Enzyme) genome editing can insert operational gene sequences into a chosen site in the genomic DNA of cells via its targetability and integrase activity. The ABBIE system offers ease of use, faster time to a readout and decreased time per genome editing experiment. SW480 and Caco-2 colon adenocarcinoma cells were used with the ABBIE system to integrate a GFP donor sequence into a safe harbor locus of the genome. Integration is to be confirmed with fluorescence microscopy and DNA sequencing. The colon cancer cell line will then be edited with the ABBIE genome editing system to include a gene cassette that overexpresses caspase genes into the genomic DNA of these cells. ABBIE-Edited colon cancer cell lines are expected to exhibit increased apoptosis as compared to non-edited controls or controls edited with DNA that do not have the caspase genes. This strategy will help lead to novel and more targetable treatments for colon cancer. Citation Format: David C. Aguilar, Alfred Gallegos. Dear ABBIE, edit that colon cancer [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 3175.

Enantioselective analysis of the pesticide imazamox after in vitro permeability study in Caco-2 cells.

Imazamox (IMX), a chiral herbicide used in cereals and oilseed crops to control weeds, is commonly sold as a racemic mixture. Its enantiomers, being chiral compounds, may exhibit unique properties when exposed to chiral environments. While IMX enantiomers have been reported to degrade differently in soil and be toxic to some species, their effects on human systems remain poorly understood. This study utilized Caco-2 (human colon adenocarcinoma cell line) cells to assess the in vitro permeability of a racemic mixture of IMX and its isolated enantiomers. Additionally, the study aimed to evaluate whether the metabolite imazamox-O-desmethyl (IMX-D) forms during the permeability process. An enantioselective chromatographic method was developed, fully validated, and the apparent permeability values were obtained. The apparent permeability of rac-IMX, (+)-IMX, and (-)-IMX was determined to be 4.15×10-5, 5.78×10-5, and 7.33×10-5cm s-1, respectively. These findings suggest that IMX exhibits high intestinal permeability, with an enantioselective absorption for (-)-IMX as compared to (+)-IMX. Finally, the permeability study in Caco-2 cells revealed that the metabolite IMX-D was not generated.

The tsRNAs (tRFdb-3013a/b) serve as novel biomarkers for colon adenocarcinomas.

The tsRNAs (tRNA-derived small RNAs) are a novel class of small non-coding RNAs derived from transfer-RNAs. Colon adenocarcinoma (COAD) is the most malignant intestinal tumor. This study focused on the identification and characterization of tsRNA biomarkers in colon adenocarcinomas. Data processing and bioinformatic analyses were performed with the packages of R and Python software. The cell proliferation, migration and invasion abilities were determined by CCK-8 and transwell assays. Luciferase reporter assay was used to test the binding of tsRNA with its target genes. With computational methods, we identified the tRNA fragments profiles within COAD datasets, and discriminated forty-two differentially expressed tsRNAs between paired colon adenocarcinomas and non-tumor controls. Among the fragments derived from the 3' end of tRNA-His-GUG (a histidyl-transfer-RNA), tRFdb-3013a and tRFdb-3013b (tRFdb-3013a/b) were notably decreased in colon and rectum adenocarcinomas, especially, tRFdb-3013a/b might tend to be down-regulated in patients with lymphatic or vascular invasion present. The clinical survival of colorectal adenocarcinoma patients with low tRFdb-3013a/b expression was significantly worse than that of high expression patients. In colon adenocarcinoma cells, tRFdb-3013a could have inhibited cell proliferations, and reduced cell migration and invasion abilities. The enrichment analyses showed that most of tRFdb-3013a correlated-genes were enriched in the extracellular matrix associated GO terms, phagosome pathway, and a GSEA molecular signature pathway. Additionally, the 3'UTR of ST3GAL1 mRNA was predicted to contain the binding site of tRFdb-3013a/b, tRFdb-3013a/b might directly target and regulate ST3GAL1 expression in colon adenocarcinomas. These results suggested that tRFdb-3013a/b might serve as novel biomarkers for diagnosis and prognosis of colon adenocarcinomas, and act a key player in the progression of colon adenocarcinomas.

Phytochemical screening and preliminary biological in vitro assessment of Camellia sinensis L. ethanolic extract

Camellia sinensis L. (Theaceae), is the most frequently consumed beverage worldwide, due to its human health benefits. The antioxidant compounds are known to act as a protective barrier of cell membranes against oxidative injury caused by various free radicals as well as by reactive oxygen species. Additionally, even phenolic compounds have pharmaceutical effects on oxidative injury from diverse causes. Therefore, the present study was conceived to establish the phytochemical screening, as well as the physicochemical profile of an ethanolic extract of aerial parts of Camelia sinensis L. acquired from a local authentic herbal distributor. In this respect, the total phenolic and flavonoid contents were investigated, as well as the antioxidant capacity of the green tea ethanolic extract obtained. For the physicochemical profile, the organic functional groups present in the green tea extract, and their thermal behavior were identified. Moreover, a preliminary in vitro biological assay was performed, through the evaluation of the antitumor potential of the green tea ethanolic extract obtained on the colon adenocarcinoma cell line (HT-29) compared to a healthy cell line, HaCaT (human immortalized keratinocytes). The results after the biological assessment suggest that the extract decreases the viability of HT-29 colon tumor cells (67% at a concentration of 100 µg/mL), showing a lesser effect on HaCaT cells; this being a first step in exploring the anticancer properties of the obtained extract.

Phytochemical analysis, antiproliferative, antibacterial, antifungal, and antiviral activities of bitter orange (Citrus aurantium) leaf essential oil

The young and old leaves of bitter orange (Citrus aurantium) were examined for their essential oil (EO) compositions, anticancer, antimicrobial, and antiviral activities. The EO yields were 0.77% in young leaves and 0.26% in old leaves. The EO composition showed the predominance of sabinene (37.17%), E-β-ocimene (8.21%), δ-3-carene (7.71%), linalool (7.35%), and limonene (5.43%) in young leaves while of sabinene (47.47%), β-sinensal (5.71%), E-β-ocimene (4.98%), δ-3-carene (4.11%), and β-caryophyllene (4.06%) in old leaves. The EOs of both young and old leaves showed potent cytotoxic activities against human lung carcinoma (A549) and colon adenocarcinoma (DLD-1) cells with IC50 < 0.1 µg/mL. The young leaf EO had higher antibacterial activity than the old leaf EO for all bacterial strains with inhibition zones (IZ) varying from 17 to 45 and 8 to 36 mm, respectively. The EOs of both young and old leaves had strong antifungal activity against all fungal strains with IZ = 90 mm. The young leaf EO was found to be the most active in protecting cells against Herpes simplex virus type-1 (HSV-1) infection during the protection, absorption, and direct periods as compared to the untreated and treated Vero cells except the replication phase.

A Novel Trypsin Kunitz-Type Inhibitor from Cajanus cajan Leaves and Its Inhibitory Activity on New Cancer Serine Proteases and Its Effect on Tumor Cell Growth.

A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617μM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24h of treatment at 70°C, after 1h treatments with different pH values, and β-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.

Doxorubicin-induced chemoresistance in Duke's type B colon adenocarcinoma cell line is aggravated in the presence of TGF-β2 through non-apoptotic cell death.

The current challenge in clinical cancer treatment is chemoresistance. Colon cells have inherently higher xenobiotic transporters expression and hence can attain resistance rapidly. Increased levels of TGF-β2 expression in patients have been attributed to cancer progression, aggressiveness, and resistance. To investigate resistance progression, we treated doxorubicin (dox) to HT-29 colon adenocarcinoma cells in the presence or absence of TGF-β2 ligand. After 1, 3-, and 7-day treatment, we investigated cell proliferation, viability, and cytotoxicity by MTT, trypan blue staining, and lactate dehydrogenase enzyme release. The mechanism of cell death was elucidated by hoechst33342 and propidium iodide dual staining and apoptosis assay. The development of resistance was detected by rhodamine123 efflux and P-glycoprotein (P-gp)/MDR1 antibody staining through fluorimetry and flow cytometry. The colony formation ability of the cells was also elucidated. Inhibition of cell proliferation was noted after day 1, while a significant reduction in viability and a significant increase in lactate dehydrogenase release was detected after day 3. Reduction of intracellular rhodamine123 levels was detected after day 3 and was significantly lower in dox with TGF-β2 treatment compared to dox alone. Increased surface P-gp levels after days 3 and 7 were observed in the treated groups. Hoechst33342/propidium iodide staining and apoptosis assay indicated non-apoptotic cell death. The cells treated with TGF-β2 had higher colony formation ability. TGF-β2 expression might play a significant role in the development of chemoresistance to doxorubicin in Duke's type B colon adenocarcinoma cell line, HT-29.

Chemical and biological investigation of Indigofera ammoxylum (DC.) Polhill. red and white phenotypes through feature-based molecular networking

Chemical investigation of ethyl acetate bark extracts of Indigofera ammoxylum red and white phenotypes led to the bio-guided isolation of four previously undescribed flavonoids, named (2S,3R)-3′,7-dihydroxy-4′,6-dimethoxyflavanol (1), (2S,3R)-6-methoxy-7-hydroxyflavanol (2), 2′,3′,7-trihydroxy-4′,6-dimethoxyisoflavone (7) and 2′,5' -dimethoxy-4′,5,7-trihydroxyisoflavanone (8), along with 14 known compounds (3–6 and 9–18). The previously undescribed structures were characterized based on NMR, HRESIMS, UV and IR data. Published spectroscopic data were used to deduce the structure of the known compounds. Eleven of the 18 isolated metabolites were evaluated for anti-inflammatory activity and cytotoxic activity against human liver carcinoma cells and human colon and colorectal adenocarcinoma cells. All tested compounds showed an anti-inflammatory activity (IC50 NO < 25 μg/mL), and compounds 2 and 3 were more selective than the positive control dexamethasone. Afromorsin (6) showed promising cytotoxic properties against both cancer cell lines (IC50 18.9 and 11.4 μg/mL). Feature-based molecular networking approach applied to bark and leaves extracts of the two phenotypes allowed to detect bioactive analogues, belonging to the families of flavones, isoflavones, flavanones, flavanols and flavonols, and to explore the chemodiversity of the species. The red and white phenotypes have a similar composition, whereas bark and leaves contain specific chemical entities. Finally, this approach highlighted a cluster of potentially bioactive and undescribed metabolites.

Comparative Solution Equilibrium Studies on Anticancer Estradiol-Based Conjugates and Their Copper Complexes

Steroids are often considered valuable molecular tools for the development of anticancer agents with improved pharmacological properties. Conjugation of metal chelating moieties with a lipophilic sterane backbone is a viable option to obtain novel anticancer compounds. In this work, two estradiol-based hybrid molecules (PMA-E2 and DMA-E2) with an (N,N,O) binding motif and their Cu(II) complexes were developed. The lipophilicity, solubility, and acid-base properties of the novel ligands were determined by the combined use of UV-visible spectrophotometry, pH-potentiometry, and 1H NMR spectroscopy. The solution speciation and redox activity of the Cu(II) complexes were also investigated by means of UV-visible and electron paramagnetic resonance spectroscopy. Two structurally analogous ligands (PMAP and DMAP) were also included in the studies for better interpretation of the solution chemical data obtained. Three pKa values were determined for all ligands, revealing the order of the deprotonation steps: pyridinium-NH+ or NH(CH3)2+, secondary NH2+, and OH. The dimethylamine derivatives (DMA-E2, DMAP) are found in their H2L+ forms in solution at pH 7.4, whereas the fraction of the neutral HL species is significant (34–37%) in the case of the pyridine nitrogen-containing derivatives (PMA-E2, PMAP). Both estradiol derivatives were moderately cytotoxic in human breast (MCF-7) and colon adenocarcinoma (Colo-205) cells (IC50 = 30–63 μM). They form highly stable complexes with Cu(II) ions capable of oxidizing ascorbate and glutathione. These Cu(II) complexes are somewhat more cytotoxic (IC50 = 15–45 μM) than their corresponding ligands and show a better selectivity profile.

Genistein Enhances TRAIL-Mediated Apoptosis Through the Inhibition of XIAP and DcR1 in Colon Carcinoma Cells Treated with 5-Fluorouracil.

Colorectal cancer is one of the most common cancers worldwide. However, surgical intervention and chemotherapy provide only limited benefits for the recovery and survival of patients. The anticarcinogenic effect of genistein has attracted attention because epidemiological studies have shown that soybean consumption is associated with a decrease in the incidence of cancer. There are limited studies on the effects of genistein in colorectal carcinoma cells. We aimed to investigate the cytotoxic, genotoxic, and apoptotic effects of genistein in SW480 and SW620 colon adenocarcinoma cells treated with 5-fluorouracil, the basis of chemotherapy, and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) ligand, the mediator of apoptosis, both alone and in combination. Cytotoxicity and genotoxicity were determined by MTT and comet assays, respectively. The apoptotic effects were evaluated by reverse transcription-polymerase chain reaction assay, with the additional use of Annexin V FITC, mitochondrial membrane potential (MMP), caspase 3, 8, and 9 activity, and reactive oxygen species (ROS) assay kits. According to our findings, genistein, 5-fluorouracil, and TRAIL had synergistic apoptotic effects because of DR5 upregulation, ROS production, and DNA damage, which were mediated by increased caspase-8, and -9 activity and decreased MMP. The applied combinations of these compounds may contribute to the resistance problem that may occur in treating colorectal cancer, with a decrease in DcR1 and XIAP genes.

A low-anticoagulant heparin suppresses metastatic dissemination through the inhibition of tumor cell-platelets association

Metastasis is the leading cause of cancer-related deaths. Despite this relevance, there is no specific therapy targeting metastasis. The interaction of the tumor cell with platelets, forming microemboli is crucial for successful hematogenous dissemination. Heparin disrupts it by a P-selectin-mediated event. However, its clinical use for this purpose is hindered by the requirement of high doses, leading to anticoagulant-related side effects. In this study, we obtained a low-anticoagulant heparin through the fractionation of a pharmaceutical bovine heparin. This derivative was referred to as LA-hep and we investigated its efficacy in inhibiting metastases and explored its capacity of suppressing the interaction between tumor cells and platelets. Our data revealed that LA-hep is as efficient as porcine unfractionated heparin in attenuating lung metastases from melanoma and colon adenocarcinoma cells in an assay with a single intravenous administration. It also prevents platelet arrest shortly after cell injection in wild-type mice and suppresses melanoma-platelets interaction in vitro. Moreover, LA-hep blocks P-selectin's direct binding to tumor cells and platelet aggregation, providing further evidence for the role of P-selectin as a molecular target. Even in P-selectin-depleted mice which developed a reduced number of metastatic foci, both porcine heparin and LA-hep further inhibited metastasis burden. This suggests evidence of an additional mechanism of antimetastatic action. Therefore, our results indicate a dissociation between the heparin anticoagulant and antimetastatic effects. Considering the simple and highly reproducible methodology used to purify LA-hep along with the data presented here, LA-hep emerges as a promising drug for future use in preventing metastasis in cancer patients.

Chemical composition, antimicrobial and antiproliferative activity of the essential oil from Ambrosia artemisiifolia L

ABSTRACT The aim of our study was to characterize the composition and certain bioactivities of the essential oil of Ambrosia artemisiifolia. Eighty organic compounds were identified in the essential oils obtained by hydrodistillation and microwave-assisted isolation. Germacrene D was the main component in both oils. The essential oil obtained by hydrodistillation exerted antibacterial activity against two S. aureus strains, with MIC values of 0.015% for the methicillin-susceptible strain and 0.25% for the methicillin-resistant strain. In the biofilm formation assay, a dose-dependent inhibition was observed in case of the K. pneumoniae ATCC 600,703. In the ethidium bromide accumulation assay, a dose-dependent inhibition activity was observed against the bacterial efflux pump of the Gram-negative bacteria E. coli ATCC 25,922. The essential oil displayed strong in vitro cytotoxicity against the multidrug-resistant (MDR) Colo 320 cancer cell line with an IC50 value of 0.0103%. The essential oil showed strong inhibition on the proliferation of the doxorubicin-sensitive Colo 205 cells (IC50 values of 0.054%) and in the case of MDR Colo 320 colonic adenocarcinoma cells (IC50 values of 0.008%). This is the first report of the cytotoxic and antiproliferative activities of the essential oil from these species.