HT-29 Human Colon Adenocarcinoma Cells Research Articles

Design, Synthesis and Biological Evaluation of 4,6-Substituted 2-Styrylquinoline Derivatives: Potential Antimicrobial and Anticancer Agents

A series of novel 4,6-substituted 2-styrylquinoline derivatives (3a-h) was designed and synthesized based on the substitution in quinoline scaffolds (with Br, H, NO2 at C6 position) as well as the substitution in the styryl moeity (at C4' position). The target compounds were investigated for their antimicrobial activity against Gram-positive Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212) and Gram-negative bacterium Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and Candida albicans (ATCC 10231) and anticancer activity against human colon adenocarcinoma HT29 cells. Compared to standard drug ciprofloxacin, compounds 3e, 3b and 3c showed significant antibacterial activity against Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. Additionally, synthesized compound 3e had significant activity against Candida albicans with MIC value of 8 µg/mL. In anticancer study, the maximum cell death rate was found in compounds 3e, 3b and 3c at the dose of 100 µM i.e., 72.9%., 44.01% and 35.46%, respectively and the measured IC50 were 58.84, 114.53 and 130 µM. The cytotoxic potential of compound 3e was confirmed by AO-EtBr dual staining assay in which red fluorescence of necrotic cells lost their membrane integrity due to toxicity induced by compound 3e. This growth inhibition and apoptotic activity of compound 3e was dose-dependent when the colour changed from green to red. Furthermore, in silico studies were conducted on the synthesized compounds to elucidate the potential mechanisms of action against dihydrofolate reductase (DHFR) from E. coli, DNA gyrase from P. aeruginosa, DHFR from S. aureus, acetolactate synthase from K. pneumoniae and DHFR from C. albicans. The derivatives also exhibited the strongest ligand-receptor binding affinity. Additionally, ADME analysis was performed to predict the pharmacokinetic profiles of the compounds, indicating good oral drug-like properties.

Role of Extracellular Vesicles in Absorption and Functional Mechanisms of Quercetin.

Quercetin (QUE), a phytochemical found in various plant foods, has been shown to have a variety of physiological activities in vivo, though biological sites where it has activities and the mechanisms of transport have not been fully elucidated. In the present study, intracellular uptake of QUE into HT-29 human colon adenocarcinoma cells is found to result in spontaneous release of extracellular vesicles (EVs), which are subsequently embedded with QUE. In addition, QUE-embedded EVs are detected in serum of QUE-administered Sprague-Dawley rats. Interestingly, the rate of cellular uptake of QUE-encapsulated EVs (EV-QUE) into RAW264.7 macrophages is markedly higher than that of free QUE. Moreover, EV-QUE suppresses lipopolysaccharide (LPS)-induced nitric oxide at a lower concentration than free QUE. The present findings suggest that QUE may be embedded in EVs in the gastrointestinal tract, then become absorbed and enter the bloodstream to exhibit biological activities.

Total Synthesis of (+)-Muricatetrocin B via a Late-Stage Co-Catalyzed Hartung-Mukaiyama Cyclization.

Convergent total synthesis of (+)-muricatetrocin B, a tetrahydrofuran-containing acetogenin with potent and selective cytotoxicity against the HT-29 human colon adenocarcinoma cell line, was achieved in 13 steps. Our synthesis is highlighted by a late-stage sequential olefin cross-metathesis/Hartung-Mukaiyama cyclization for convergent assembly of the 2,5-trans-substituted tetrahydrofuran ring.

Beyond Antioxidant Activity: Redox Properties of Catechins May Affect Changes in the DNA Methylation Profile-The Example of SRXN1 Gene.

The role of catechins in the epigenetic regulation of gene expression has been widely studied; however, if and how this phenomenon relates to the redox properties of these polyphenols remains unknown. Our earlier study demonstrated that exposure of the human colon adenocarcinoma HT29 cell line to these antioxidants affects the expression of redox-related genes. In particular, treatment with (-)-epigallocatechin (EGC) downregulated transcription of gene encoding sulfiredoxin-1 (SRXN1), the peroxidase involved in the protection of cells against hydrogen peroxide-induced oxidative stress. The aim of this study was to investigate whether the observed SRXN1 downregulation was accompanied by changes in the DNA methylation level of its promoter and, if so, whether it was correlated with the redox properties of catechins. The impact on DNA methylation profile in HT29 cells treated with different concentrations of five catechins, varying in chemical structures and standard reduction potentials as well as susceptibility to oxidation, was monitored by a methylation-sensitive high-resolution melting technique employing the SRXN1 promoter region as a model target. We demonstrated that catechins, indeed, are able to modulate DNA methylation of the SRXN1 gene in a redox-related manner. The nonlinear method in the statistical analysis made it possible to fish out two parameters (charge transfer in oxidation process Qox and time of electron transfer t), whose strong interactions correlated with observed modulation of DNA methylation by catechins. Based on these findings, we present a proof-of-concept that DNA methylation, which limits SRXN1 expression and thus restricts the multidirectional antioxidant action of SRXN1, may represent a mechanism protecting cells against reductive stress caused by particularly fast-reacting reductants such as EGC and (-)-epicatechin gallate (ECG) in our study.

A Novel ssDNA Aptamer Targeting Carcinoembryonic Antigen: Selection and Characterization.

Simple SummaryCarcinoembryonic antigen (CEA), a well-known cancer biomarker that is used to diagnose various cancers, notably colorectal cancer, was used as a target to select and characterize single-stranded DNA aptamers. These short and compact nucleic acid sequences, known as aptamers, are becoming ever more popular in both therapeutics and diagnostics. In this study, SELEX and NGS techniques were employed in the aptamer selection processes. Aptamer characterization techniques, such as enzyme-linked oligonucleotide assay (ELONA), dot blot, and bioinformatics assays, were conducted to observe the affinity binding of the selected aptamer to the target protein. Confocal microscopy results demonstrated how the fluorescently labelled aptamer sequence recognized CEA, which is expressed in HT-29 human colon adenocarcinoma cell line. These findings show that the selected aptamer has the potential to be employed as a recognition component in rapid detection systems, such as aptasensors.One of the major causes of a drastically shorter life expectancy and one of the most prevalent diseases in the world today is cancer. Given the data on the rise in cancer cases throughout the world, it is obvious that, despite the diagnostic techniques currently being used, there is a pressing need to develop precise and sensitive techniques for early diagnosis of the disease. A high degree of affinity and specificity towards particular targets is maintained by the short nucleic acid molecules known as aptamers. Aptamers outperform antibodies due to their unique benefits, such as their simplicity in synthesis and modification, lack of toxicity, and long-term stability. Utilizing an accurate recognition element and a robust signal transduction mechanism, molecular diagnostics can be extremely sensitive and specific. In this study, development of new single-stranded DNA aptamers against CEA for use in cancer diagnostics was accomplished using SELEX and NGS methods. As a result of 12 iterative SELEX rounds, nine aptamer candidates against CEA were developed. NGS comparative analysis revealed that round twelve had an enriched number of aptamers that were specifically bound, as opposed to round eight. Among the selected nine sequences characterized by bioinformatics analysis and ELONA, an aptamer sequence with the highest specificity and affinity for the target protein was identified and further examined. Aptamer sequence (6) was screened in a concentration-dependent assay, specificity analysis was performed, and its potential secondary and tertiary structures were predicted, which enabled us to test one of the possible putative interactions with CEA. Finally, aptamer sequence (6) labelled with a Cy5 fluorescent tag was used in confocal microscopy to observe its binding towards the CEA expressed in HT-29 human colon adenocarcinoma cell line.

Ethyl acetate partition obtained from the methanol extract of Muntingia calabura leaf exerts effective in vitro antiproliferative activity against the HT-29 colon cancer

Methanol extract of Muntingia calabura L. leaf (MEMCL) has been shown to exert the antiproliferative activity against the HT-29 (human colon adenocarcinoma) cell line. To further investigate on the medicinal potential of this plant, MEMCL was sequentially partitioned to obtain the petroleum ether, ethyl acetate and aqueous partitions, which was then tested against the HT-29 cell line and also subjected to the in vitro anti-inflammatory study. The most effective partition was also subjected to the phytoconstituents analysis using the UHPLC-ESI-MS. Findings showed that the ethyl acetate partition (EAP) exerts the most effective antiproliferative activity (IC50 = 58.0±12.9 μg/mL) without affecting the 3T3 normal fibroblast cells, exhibits the highest anti-inflammatory effect when assessed using the lipoxygenase (> 95%) and xanthine oxidase (> 70%) assays, and contained various types of polyphenolics. In conclusion, M. calabura exerts apoptotic-mediated antiproliferative activity, partly via the anti-inflammatory action and synergistic action between the polyphenolics.

Influence of different food models and storage temperatures on the bacterial growth inhibition by maltodextrin laurate and sucrose laurate and investigation of their cytotoxicity

Maltodextrin esterified to laurate (ML) was synthesized enzymatically and compared to commercial sucrose laurate (SL) for microbial growth-inhibitory properties in food systems. There was broad microbial inhibition by ML in growth media, but practical information about various foods and storage temperatures is limited with no toxicological information. This research investigated the antimicrobial property of ML against two spoilage bacteria (Bacillus subtilis and Lactobacillus plantarum) in growth media and related food models stored at different temperatures (4 °C, 22 °C, and 35 °C). Additionally, in vitro cytotoxicity of ML was investigated in human colon adenocarcinoma HT-29 cells and human normal colon epithelial CCD 841 CoN cells using a standard colorimetric assay. ML was highly inhibitive against L. plantarum in apple juice at all temperatures tested. SL could inhibit both bacteria in the food models tested (milk, apple juice, tomato ketchup, French dressing) at 4 °C. ML was not observed to be cytotoxic to either cell line up to 500 μg/mL, whereas SL was evidently cytotoxic with IC50 ranging from 84 to 94 μg/mL. In conclusion, ML can be an alternative to commercial SL as a safe food additive in certain products.

Elucidation of potential anticancer, antioxidant and antimicrobial properties of some new triazole compounds bearing pyridine-4-yl moiety and cyclobutane ring

Thiosemicarbazides (2a–e) were obtained by the interaction of pyridine-4-carboxylic acid hydrazide (1) with five different isothiocyanate (RNCS) derivatives. By addition of KOH to the reaction medium, ethyl, allyl, phenyl and benzyl, p-tolyl substituted 1,2,4-triazoles (3a–e) were obtained. 3a–e were dissolved in dry acetone containing K2CO3 in the presence of 2-chloro-1-(3-methyl-3- mesitylcyclobutyl) ethanone (4) to give 3,4,5-trisubstituted 1,2,4-triazole sulfanyl compounds containing a cyclobutane ring (5a–e). The structures of the final compounds were confirmed by elemental analyses, FT-IR, 1H NMR and 13C NMR. Due to many activities regarding pharmacology, Compounds such as 1,2,4-triazole attract high attention in the fields of chemistry, pharmacology, and biology. All the newly synthesised compounds were screened for their Antioxidant, antimicrobial, anti-cancer activities. The measurement of the antioxidant feature of the compounds was performed by DPPH radical scavenging activity technique. It was observed that the activity of some of the compounds was close to the standard antioxidant BHT. Furthermore, the anticancer effects of compounds were investigated against HT29 human colon adenocarcinoma cells. It has been noted that all -compounds inhibited cancerous cells in a statistically significant compared to the control. For the measurement of antimicrobial activity Agar well diffusion method was preferred. For the antimicrobial study, S. aureus, E. faecalis, P. aeruginosa, E. coli, and a single fungi C. albicans have been used. They could also be stated as ATCC 29213, 29212, 27853, 2592, and 10231 respectively. The resulting range of MIC values for the chemicals was between 15.625 - >125 µM. In conclusion; all compounds have shown various and important bioactivities at different levels, such as being antioxidant, antimicrobial, and anticancer.

Regulation of apoptosis and autophagy by albendazole in human colon adenocarcinoma cells

Albendazole (ABZ) was initially introduced as an anthelmintic, however, many studies have reported with its anticancer effects. We investigated the anti-tumor effects of ABZ in vitro in human colon adenocarcinoma HCT-15, HCT-116, HT-29, and SW480 cell lines in this study. The cytotoxicity of ABZ was analyzed in colon adenocarcinoma cell lines and normal CCD18Co cells. We found that ABZ induced the subG1 arrest during cell cycle progression, increased the late apoptotic cells, shifted of peak TUNEL-labeled cells peak, and induced apoptosis. Then effects on autophagy activation was confirmed by acridine orange (AO), MDC staining, and immunocytochemistry of LC3. It was observed that ABZ can induce the autophagy activation through modulating the levels of LC3, Atg7, and beclin-1. For mechanistic studies, apoptosis blocker (Z-DEVD-FMK) and autophagy inhibitor (3-MA) were used to confirm that whether ABZ has apoptosis and autophagy specific effects, and reversal in both these cell death processes were noted. The effects of ABZ on AMPK, MAPKs, and ULK induction was also evaluated. We noticed that N-acetyl cysteine (NAC), a broad spectrum antioxidant, can effectively inhibit both apoptosis and autophagy. However, ABZ could even recover suppression of apoptosis and autophagy caused by NAC in colon cancer cells. Therefore, ABZ can potentially up-regulate both the apoptosis and autophagy to significantly suppress tumorigenesis in colorectal cancer cell lines.

Structure-performance relationships of four lysosomal markers used for the imaging of HT-29 cancer cells and a cellular model of lysosomal storage disease (Niemann-Pick C)

Four new BODIPY derivatives with a potential tendency to aggregation have been synthesized and characterized by means of NMR techniques, mass spectrometry and UV–Vis/fluorescence spectroscopies. The objective of this study is to determine which structural factors of the new molecules influence most notably the cellular uptake, intracellular location and fluorescence imaging abilities. The behaviour of the compounds in organic solvent and aqueous solution has been studied. In organic solvents (DMSO, ethanol and toluene), the photophysical properties of the new molecules are almost independent of the building blocks used to synthesize the pendant moieties (non-fluorogenic parts). In an aqueous environment (HEPES Buffer Solution, pH 7), at 10 μM, three of the compounds (1, 2 and 4) tend to form weakly emissive nanoparticles (DLS determination) whereas one of them (3) remains soluble and highly fluorescent. In the nanomolar range of concentration, all the compounds are aqueous soluble. The cellular internalisation of the compounds (10 nM) has been studied in human colon adenocarcinoma HT-29 cells by means of flow cytometry and confocal laser scanning microscopy. All the compounds were uptaken by HT-29 cells, but notably molecule 3 (made with lysine as a building block) was the one displaying a higher loading and a more clear lysosomal location (0.88 Pearson's correlation coefficient in colocalization assays using lysosomal fluorescent probe LysoTracker DND-99). Molecule 3 also performed better than the valine derivative 1 as a lysosomal marker in a cellular model (human adrenal carcinoma SW13/cl.2 cells) of lysosomal storage disease (Niemann-Pick type C).

In vitro anticancer activity of ethanolic extract of Stoechospermum marginatum against HT-29 human colon adenocarcinoma cells

Colorectal cancer is a one of the leading causes of death globally and its clinical management of cancer involves chemotherapy. Increase in the development of resistance to the drugs used in the cancer treatment and serious side effects associated with chemotherapeutic drugs are the major limitations in cancer therapy. Hence, there exists a huge need to develop safer natural therapeutic products for cancer therapy. In this study, ethanolic extract of Stoechospermum marginatum was evaluated for its anticancer activity. The cytotoxicity of S. marginatum extract was evaluated on HT-29 cells by MTT assay. Trypan blue cell viability was also carried out to evaluate cytotoxicity and antiproliferative effect. The apoptosis-inducing potential of the extract was analyzed by acridine orange and ethidium bromide dual staining method, mitochondrial membrane potential assay and FITC Annexin V-Propidium iodide staining method. The ethanolic extract of S. marginatum showed significant dose-dependent cytotoxicity in HT-29 cells Treatment with S. marginatum extract increased number of apoptotic cells in HT-29 cells and caused damage to mitochondrial membrane potential. The findings of the present study confirmed in vitro anticancer activity of ethanolic extract S. marginatum

Effects of bisphenol A on the proliferation, migration, and tumor growth of colon cancer cells: In vitro and in vivo evaluation with mechanistic insights related to ERK and 5-HT3

Bisphenol A (BPA) is a well-known endocrine-disrupting chemical related to the carcinogenesis of estrogen-responsive organs. Although human exposure to BPA mainly occurs via the oral route, its association with colon cancer has not been fully elucidated. We investigated the effects of BPA on the proliferation, migration, and tumor growth of colon cancer cells. BPA significantly promoted the proliferation of HT-29 human colon adenocarcinoma cells in a time- and dose-dependent manner. BPA also increased HT-29 cells migration. BPA increased the phosphorylation of extracellular signal-regulated kinase (ERK), and inhibition of the ERK pathway attenuated BPA-induced proliferation and migration. In addition, BPA reduced E-cadherin expression, a key factor impeding epithelial-to-mesenchymal transition, and increased 5-HT3 receptors expression, a major mitogenic factor. In xenograft models, tumor volume of the BPA-treated nude mice was 4.6 times that of the saline-treated group. Our findings provide primary evidence regarding the link between BPA and human colon cancer by demonstrating that BPA promotes the proliferation, migration, and tumor growth of colon cancer cells in both in vitro and in vivo models. In addition, we provided the mechanism of action of BPA, involved in the activation of the ERK pathway, the decrease in E-cadherin, and the increase in 5-HT3 receptors.

Synthesis, Characterization and Biological Evaluation of Novel Triazolyl - Acridine Derivatives as Cytotoxic Agents

Acridine and Triazols both are biologically active heterocyclic rings with cytotoxic potential. Triazolyl- acridine adduct attract the attention in the field of medicinal chemistry. Here we synthesized a series of triazolyl- acridine compounds by the appropriate procedures. Structure of all synthesized compounds was confirmed by the various spectroscopic methods e.g. FT-IR, proton NMR and Mass spectrograms. All synthesized triazolyl-acirdines compounds were assayed in-vitro for cytotoxic activity against MCF-7 (human breast adenocarcinoma cell line) and HT-29 (human colon adenocarcinoma cell line) cells by MTT- assay. All target compounds shows increasing activity in dose dependent manner. However MPSP-9 was sensitive against MCF-7 but compound MPSP-1 was most sensitive with minimum inhibitory concentration (IC50 value) against MCF-7 and HT-29 both cell lines.

Development of enteric-coated, biphasic chitosan/HPMC microcapsules for colon-targeted delivery of anticancer drug-loaded nanoparticles

Oral delivery of anticancer drug-loaded nanoparticles (NPs) to the colon offers opportunities to improve colorectal cancer (CRC) treatment by increasing the free drug concentration at tumour sites and/or enhancing NP accumulation in tumours. Indomethacin, 5-FU and curcumin, were entrapped separately in Eudragit RS NPs (approximately 10% w/w loading) using nanoprecipitation and incorporated in biphasic chitosan/HPMC microcapsules (MCs) using aerosolisation. The MCs were designed to release NPs primarily in the colon following chitosan breakdown by bacterial enzymes. Around 10% of the drug-loaded NPs was released from MCs in simulated intestinal fluid (SIF) in 6 h and 20% in simulated colon fluid (SCF). Indomethacin release from MCs was absent in simulated gastric fluid (SGF) and restricted to around 10% in SIF and SCF, respectively, demonstrating potential for delivering a large fraction of contained drug to the colon. Curcumin release from NPs or NP-loaded MCs was negligible in SGF, SIF and SCF, revealing opportunities for delivery of curcumin-loaded NPs to the colon for accumulation in tumours. Curcumin-loaded NPs reduced proliferation of human colon adenocarcinoma HT-29 cells by 83% compared with 50% for free curcumin. These findings demonstrate the potential of chitosan/HPMC microcapsules as a colon-specific delivery vehicle for oral nanomedicines directed against colorectal cancer.

Novel N-Substituted Amino Acid Hydrazone-Isatin Derivatives: Synthesis, Antioxidant Activity, and Anticancer Activity in 2D and 3D Models In Vitro

A series of novel mono and bishydrazones each bearing a 2-oxindole moiety along with substituted phenylaminopropanamide, pyrrolidin-2-one, benzimidazole, diphenylmethane, or diphenylamine fragments were synthesized, and their anticancer activities were tested by MTT assay against human melanoma A375 and colon adenocarcinoma HT-29 cell lines. In general, the synthesized compounds were more cytotoxic against HT-29 than A375. 3-((4-Methoxyphenyl)(3-oxo-3-(2-(2-oxoindolin-3-ylidene)hydrazinyl)propyl)amino)-N′-(2-oxoindolin-3-ylidene)propanehydrazide and (N′,N‴)-1,1′-(methylenebis(4,1-phenylene))bis(5-oxo-N′-(2-oxoindolin-3-ylidene)pyrrolidine-3-carbohydrazide) were identified as the most active compounds against HT-29 in 2D and 3D cell cultures. The same compounds showed the highest antioxidant activity among the synthesized compounds screened by ferric reducing antioxidant power assay (FRAP). Their antioxidant activity is on par with that of a well-known antioxidant ascorbic acid.

Synthesis and cytotoxic activity of organotin(IV) diallyldithiocarbamate compounds as anticancer agent towards colon adenocarcinoma cells (HT-29).

ContextDiphenyltin(IV) diallyldithiocarbamate compound (Compound 1) and triphenyltin(IV) diallyldithiocarbamate compound (Compound 2) are two newly synthesised compounds of organotin(IV) with diallyldithiocarbamate ligands.ObjectiveTo assess the cytotoxic effects of two synthesised compounds against HT-29 human colon adenocarcinoma cells and human CCD-18Co normal colon cells.Materials and methodsTwo successfully synthesised compounds were characterised using elemental (carbon, hydrogen, nitrogen, and sulphur) analysis, Fourier-Transform Infrared (FTIR), and 1H, 13C 119Sn Nucleus Magnetic Resonance (NMR) spectroscopies. The single-crystal structure of both compounds was determined by X-ray single-crystal analysis. The cytotoxicity of the compounds was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazholium bromide (MTT) assay upon 24 h of treatment. While the mode of cell death was determined based on the externalisation of phosphatidylserine using a flow cytometer.ResultsThe elemental analysis data of the two compounds showed an agreement with the suggested formula of (C6H5)2Sn[S2CN(C3H5)2]2 for Compound 1 and (C6H5)3Sn[S2CN(C3H5)2] for Compound 2. The two major peaks of infrared absorbance, i.e., ν(C = N) and ν(C = S) were detected at the range of 1475–1479 cm−1 and 972–977 cm−1, respectively. The chemical shift of carbon in NCS2 group for Compound 1 and 2 were found at 200.82 and 197.79 ppm. The crystal structure of Compound 1 showed that it is six coordinated and crystallised in monoclinic, P21/c space group. While the crystal structure of Compound 2 is five coordinated and crystallised in monoclinic, P21/c space group. The cytotoxicity (IC50) of the two compounds against HT-29 cell were 2.36 μM and 0.39 μM. Meanwhile, the percentage of cell death modes between 60% and 75% for compound 1 and compound 2 were mainly due to apoptosis, suggesting that both compounds induced growth arrest.ConclusionOur study concluded that the synthesised compounds showed potent cytotoxicity towards HT-29 cell, with the triphenyltin(IV) compound showing the highest effect compared to diphenyltin(IV).

Piperine and Celecoxib synergistically inhibit colon cancer cell proliferation via modulating Wnt/β-catenin signaling pathway

BackgroundCelecoxib (CXB), a selective COX-2 inhibitor NSAID, has exhibited prominent anti-proliferative potential against numerous cancers. However, its low bioavailability and long term exposure related cardiovascular side effects, limit its clinical application. In order to overcome these limitations, natural bioactive compounds with lower toxicity profile are used in combination with therapeutic drugs. Therfore, in this study Piperine (PIP), a natural chemo-preventive agent possessing drug bioavailability enhancing properties, was considered to be used in combination with low doses of CXB. PurposeWe hypothesized that the combination of PIP with CXB will have a synergistic anti-proliferative effect on colon cancer cells. Study designThe potency of PIP and CXB alone and in combination was evaluated in HT-29 human colon adenocarcinoma cells and mechanism of growth inhibition was investigated by analyzing the players in apoptotic and Wnt/β-catenin signaling pathways. MethodsThe effect of PIP on the oral bioavailability of CXB in mice was investigated using HPLC analysis. The study investigated the synergistic anti-proliferative effect of CXB and PIP on HT-29 cells and IEC-6 non-tumorigenic rat intestinal epithelial cells by SRB cell viability assay. Further, the cellular and molecular mechanism(s) involved in the anti-proliferative combinatorial effect was extensively explored in HT-29 cells by flow cytometry and western blotting. The in vivo efficacy of this combination was studied in CT26.WT tumor syngeneic Balb/c mice model. ResultsPIP as a bioenhancer increased the oral bioavailability of CXB (129%). The IC50 of CXB and PIP were evaluated to select doses for combination treatment of HT-29 cells. The drug combinations having combination index (CI) less than 1 were screened using CompuSyn software. These combinations were significantly cytotoxic to HT-29 cells but IEC-6 were least effected. Further, the mechanism behind CXB and PIP mediated cell death was explored. The co-treatment led to reactive oxygen species generation, mitochondrial dysfunction, caspase activation and enhanced apoptosis in HT-29 cells. Additionally, the combination treatment synergistically modulated Wnt/β-catenin pathway, downregulated the stemness markers and boosted therapeutic response in CT26 syngeneic Balb/c mice. ConclusionThe outcomes of the study suggests that combining CXB and PIP offers a novel approach for the treatment of colon cancer.

Induction and Detection of Necroptotic Cell Death in Mammalian Cell Culture.

The study of necroptosis is a rapidly growing field in current research of cell death mechanisms and cancer treatment strategies. While apoptotic cells can be reliably identified via annexin V assay, necroptosis is not associated with exposure of easily detectable markers. The most reliable way to identify necroptotic events is immunochemical detection of active phosphorylated RIPK1, RIPK3, and MLKL proteins facilitating necroptosis execution. This chapter describes a detailed protocol on necroptosis induction in human colon adenocarcinoma HT-29 cells, preparation of various positive and negative controls, detection of necroptosis mediator proteins via Western Blot analysis, and interpretation of results. This protocol allows reliable and specific detection of necroptosis in cell culture or tissue samples, and it provides a well-established model suitable for detailed studies of necroptosis molecular mechanisms in vitro.

Chemical constituents and biological activities of African medicinal tree Sterculia setigera Delile stem bark

Stem bark of Sterculia setigera Delile, family Sterculiaceae, is used in traditional medicine in Sudan and other African countries to treat various diseases. This study aimed to investigate the chemical constituents, antioxidant, enzyme inhibition and antiproliferative activities of stem bark extracts and an isolated compound. Compounds were extracted using hexane and dichloromethane: ethanol (DE) (1:1). Chromatographic techniques were performed to characterize the phenolic compounds. We isolated lupeol as a bioactive compound in S. setigera from DE extract. The antioxidant and enzyme inhibitory properties of each extract were evaluated. The antiproliferative activity of the DE extract was determined. Results showed that five known compounds namely; procyanidins as dimer B, trimer C1 and tetramer, 3,4-dimethoxyphenol ß-D-apiofuranosyl (1″→6″)- ß -D-glucopyranoside and (+)- catechin were identified or tentatively elucidated, in addition to the isolation and characterization of lupeol. DE extract showed significantly (p<0.05) higher antioxidant activity than that observed from the hexane extract. Both extracts displayed considerable enzyme inhibitory activity. DE extract revealed remarkable tyrosinase inhibitory activity (115.26±1.10 mg kojic acid equivalents (KAE)/g) and displayed high antiproliferative activity against human colon adenocarcinoma HT29 (IC50 4.4 ± 0.8 µg/ml) and HCT116 (IC50 7.3 ± 2.2 µg/ml) cell lines without any toxicity to Vero normal cells. Isolated lupeol exerted high tyrosinase inhibitory (37.68±5.65 mg KAE/g) and α-amylase (27.64±0.55 mg galanthamine equivalents/g) activities. In silico study showed that 3,4-Dimethoxyphenol ß -D-apiofuranosyl (1″→6″)-ß-D-glucopyranoside revealed the best docking score for tyrosinase. In conclusion, stem bark of S. setigera could be a rich natural sources of bioactive agents.

Macromolecular design of folic acid functionalized amylopectin–albumin core–shell nanogels for improved physiological stability and colon cancer cell targeted delivery of curcumin

Nanogels have potential for encapsulating cancer therapeutics, yet their susceptibility to physiological degradation and lack of cellular specificity hinder their use as effective oral delivery vehicles. Herein, we engineered novel albumin-core with folic acid functionalized hyperbranched amylopectin shell-type nanogels, prepared through a two-step reaction and loaded with curcumin while the proteinaceous core was undergoing thermal gelation. The nanogels had a mean hydrodynamic diameter of ca. 90 nm and ζ-potential of ca. −24 mV. Encapsulation of curcumin within the nanogels was restored, up to ca. 0.05 mg mL−1, beyond which, a gradual increase in size and a decrease in ζ-potential was observed. The core–shell structures were resilient to in vitro physiological oral-gastrointestinal digestion owing to a liquid crystalline B- and V-type polymorphism in the polysaccharide shell, the latter being driven by the shell functionalization with folic acid. Additionally, these biocompatible nanogels restored stability of the encapsulated curcumin and exhibited augmented cellular uptake and retention specifically in folate receptor-positive HT29 human colon adenocarcinoma cells, inducing early-stage apoptosis. Novel insights from this study represent a promising platform for rational designing of future oral delivery systems that can surmount physiological barriers for delivering cancer therapeutics to colon cancer cells with improved stability and specificity.