HCT116 Research Articles

Anticancer Effect by Combined Treatment of Artemisia annua L. Polyphenols and Docetaxel in DU145 Prostate Cancer Cells and HCT116 Colorectal Cancer Cells.

Docetaxel (DTX), a semi-synthetic analogue of paclitaxel (taxol), is known to exert potent anticancer activity in various cancer cells by suppressing normal microtubule dynamics. In this study, we examined how the anticancer effect of DTX is regulated by polyphenols extracted from Korean Artemisia annua L. (pKAL) in DU145 prostate cancer cells (mutant p53) and HCT116 colorectal cancer cells (wild-type p53). Here, we show that the anticancer effect of DTX was enhanced more significantly by pKAL in HCT116 cells than in DU145 cells via phase-contrast microscopy, CCK-8 assay, Western blot, and flow cytometric analysis of annexin V/propidium iodide-stained cells. Notably, mutant p53 was slightly downregulated by single treatment of pKAL or DTX in DU145 cells, whereas wild-type p53 was significantly upregulated by pKAL or DTX in HCT116 cells. Moreover, the enhanced anticancer effect of DTX by pKAL in HCT116 cells was significantly associated with the suppression of DTX-induced p53 upregulation, increase of DTX-induced phospho-p38, and decrease of DTX-regulated cyclin A, cyclin B1, AKT, caspase-8, PARP1, GM130, NF-κB p65, and LDHA, leading to the increased apoptotic cell death and plasma membrane permeability. Our results suggest that pKAL could effectively improve the anticancer effect of DTX-containing chemotherapy used to treat various cancers expressing wild-type p53.

Identification of new 5-(2,6-dichlorophenyl)-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-7-carboxylic acids as p38α MAPK inhibitors: Design, synthesis, antitumor evaluation, molecular docking and in silico studies

In pursuit of discovering novel scaffolds that demonstrate potential inhibitory activity against p38α MAPK and possess strong antitumor effects, we herein report the design and synthesis of new series of 17 final target 5-(2,6-dichlorophenyl)-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-7-carboxylic acids (4–20). Chemical characterization of the compounds was performed using FT-IR, NMR, elemental analyses and mass spectra of some representative examples. With many compounds showing potential inhibitory activity against p38α MAPK, two derivatives, 8 and 9, demonstrated the highest activity (>70 % inhibition) among the series. Derivative 9 displayed IC50 value nearly 2.5 folds more potent than 8. As anticipated, they both showed explicit interactions inside the kinase active site with the key binding amino acid residues. Screening both compounds for cytotoxic effects, they exhibited strong antitumor activities against lung (A549), breast (MCF-7 and MDA MB-231), colon (HCT-116) and liver (Hep-G2) cancers more potent than reference 5-FU. Their noticeable strong antitumor activity pointed out to the possibility of an augmented DNA binding mechanism of antitumor action besides their kinase inhibition. Both 8 and 9 exhibited strong ctDNA damaging effects in nanomolar range. Further mechanistic antitumor studies revealed ability of compounds 8 and 9 to arrest cell cycle in MCF-7 cells at S phase, while in HCT-116 treated cells at G0-G1 and G2/M phases. They also displayed apoptotic induction effects in both MCF-7 and HCT-116 with total cell deaths more than control untreated cells in reference to 5-FU. Finally, the compounds were tested for their anti-migratory potential utilizing wound healing assay. They induced a significant decrease in wound closure percentage after 24 h treatment in the examined cancer cells when compared to untreated control MCF-7 and HCT-116 cells better than 5-FU. In silico computation of physicochemical parameters revealed the drug-like properties of 8 and 9 with no violation to Lipinski’s rule of five as well as their tolerable ADMET parameters, thus suggesting their utilization as potential future drug leads amenable for further optimization and development.

Structurally diverse cucurbitane-type triterpenoids from the tubers of Hemsleya chinensis with cytotoxic activity

Ten previously undescribed cucurbitane-type triterpenoids, namely hemslyencins A−F (1–6) and hemslyencosides A−D (7−10), together with twenty previously reported compounds (11–30), were isolated from the tubers of Hemsleya chinensis. Their structures were elucidated by unambiguous spectroscopic data (UV, IR, HR-ESI-MS, 1D and 2D NMR data). Hemslyencins A and B (1 and 2) possessing unique 9, 11-seco-ring system with a six-membered lactone moiety, were the first examples among of the cucurbitane-type triterpenoids, and hemslyencins C and D (3 and 4) and hemslyencoside D (10) are the infrequent pentacyclic cucurbitane triterpenes featuring a 6/6/6/5/6 fused system. The cytotoxic activities of all isolated compounds were evaluated against MCF-7, HCT-116, HeLa, and HepG2 cancer cells, and their structure-activity relationships (SARs) was discussed as well. Compounds 17, 25, and 26 showed significant cytotoxic effects with IC50 values ranging from 1.31 to 9.89 μM, among which compound 25 induced both apoptosis and cell cycle arrest at G2/M phase in a dose dependent manner against MCF-7 cells.

A37 POSTBIOTIC BACTERIAL MEMBRANE VESICLES FROM B. SUBTILIS DELIVER NOD2 LIGANDS AND PROMOTE IN VITRO WOUND RE-EPITHLIALIZATION THROUGH RIPK2 SIGNALING

Abstract Background Bacterial membrane vesicles (MVs) are bilipid nanoparticles secreted as a conserved method of intercellular communication. MVs from Gram-positive bacteria carry diverse bacterial products and represent a unique opportunity in the growing field of postbiotics. This native machinery for mediating microbe-host interactions makes MVs a promising tool for intestinal drug delivery in the context of Crohn’s disease (CD). CD is a chronic inflammatory bowel disease characterized by relapsing inflammation of the digestive tract. CD is associated with (1) loss of function mutations in the host gene encoding the nuclear-binding oligomerization domain 2 (NOD2) pattern recognition receptor and (2) a gut microbiome with reduced capabilities to generate NOD2 stimulating muropeptides from peptidoglycan. Nod2-/- mice have decreased barrier integrity and impaired epithelial restitution upon challenge. However, there are currently no therapeutic strategies targeting NOD2 for CD management. Aims The aims of this study were to (1) leverage MVs as a biological system for NOD2 ligand delivery to intestinal epithelial cells and (2) determine the roll of probiotic MVs and bacterial ligand synergy in wound re-epithelialization. Methods MVs were purified from B. subtilis through filtration and ultracentrifugation. MVs were characterized through nanoparticle tracking analysis using NanoSight300. WT and NOD2-/- HCT116 cells, a human colorectal carcinoma cell line, were utilized for IL-8 secretion quantification by ELISA, gene expression by qPCR, and in vitro scratch wound assay by Incucyte quantification. Results Here, we demonstrate for the first time that MVs from B. subtilis deliver NOD2 ligands in vitro. Treatment with isolated MVs induces IL8 secretion and CXCL1 expression in a NOD2-dependent manner. Furthermore, MVs promote re-epithelialization after in vitro scratch wounding. This effect was completely blunted by the addition of an inhibitor for RIPK2, a downstream transducer required for NOD2 signaling. Through bacterial pathogen-associated molecular patterns (PAMPs) screening, we found that wound re-epithelialization is promoted by PAMP synergy but dependent on RIPK2 signaling. Conclusions This work suggests that delivery of muropeptides by probiotic-derived MVs is a promising strategy to promote epithelial regeneration during injury through targeting NOD2. Future directions will work towards validating these findings in human ileal primary cells and murine models of intestinal injury. Funding Agencies CIHRMedicine by Design

A26 BIOLOGICAL CHARACTERIZATION OF THE INTERACTION BETWEEN SIRT1 AND HNF4Α2 IN INTESTINAL EPITHELIAL CELLS

Abstract Background Colorectal cancer (CRC) is the third most common cancer in Canada. HNF4A locus is amplified in CRC, while additional reports suggest that hepatocyte nuclear factor 4 alpha (HNF4α) is associated with increased proliferation and disease development. This dual role as a tumor suppressor or oncoprotein might be due to the production of 12 spliced isoforms with structural differences and variable tissue expression. It suggests distinct functions for each isoform according to their specific interaction complexes. However, little is known about the nature of these protein complexes and their biological functions during intestinal physiopathology. Recently, using a BioID2 and mass spectrometry approach, our laboratory showed a possible interaction between HNF4α2 and sirtuin 1 (SIRT1) in intestinal epithelial cells from a colon carcinoma (HCT116). HNF4α2 is one of the most potent isoforms in the control of transcriptional activity of multiple intestinal epithelial genes related to regulating cell death, angiogenesis, apoptosis, response to injury, and response to drugs, among others. Aims To characterize the possible interaction between HNF4α2 and SIRT1 in intestinal epithelial cells at the biological level. Methods We performed SIRT1 knockdown using shRNAs in HCT116 cells expressing the HNF4α2 isoform in an inducible manner by doxycycline (DOX). Subsequently, we conducted a proximity ligation assay to validate the interaction between HNF4α2 and SIRT1 in HCT116 cells. Additionally, protein purification was performed to validate their physical interaction through EMSA. A transcriptome analysis was carried out using RNA-seq to define the biological processes involved in the interaction between the two proteins. Results We observed an interaction signal between HNF4α2 and SIRT1 in HCT116 cells, similar to that observed for IRFBB2, whose interaction with HNF4α2 has been previously validated. This signal decreased when cells were treated with shRNA for SIRT1 and was not observed in cells not induced by DOX. EMSA analyses revealed a direct interaction signal between HNF4α2 and SIRT1. RNA-seq analyses showed a loss in the regulation of over 95% of genes targeted by HNF4α2 when cells were treated with shRNA for SIRT1. An enrichment analysis for GO annotations using ShyniGO v. 0.77 software showed an enrichment of genes for biological processes such as apoptosis, inflammation, cell migration, cell invasion, cell death, and various cancer-related signaling pathways. Conclusions SIRT1 displays physical interaction with HNF4α2 and significantly affects the transcriptional regulation of this isoform in the context of epithelial cells. Their interaction is involved in processes related to intestinal inflammation and cancer progression. Pharmacological targeting of SIRT1 could represent a viable strategy in treating CRC and other intestinal diseases Funding Agencies CIHRNSERC

Cone photoreceptor phosphodiesterase PDE6H inhibition regulates cancer cell growth and metabolism, replicating the dark retina response

BackgroundPDE6H encodes PDE6γ′, the inhibitory subunit of the cGMP-specific phosphodiesterase 6 in cone photoreceptors. Inhibition of PDE6, which has been widely studied for its role in light transduction, increases cGMP levels. The purpose of this study is to characterise the role of PDE6H in cancer cell growth.MethodsFrom an siRNA screen for 487 genes involved in metabolism, PDE6H was identified as a controller of cell cycle progression in HCT116 cells. Role of PDE6H in cancer cell growth and metabolism was studied through the effects of its depletion on levels of cell cycle controllers, mTOR effectors, metabolite levels, and metabolic energy assays. Effect of PDE6H deletion on tumour growth was also studied in a xenograft model.ResultsPDE6H knockout resulted in an increase of intracellular cGMP levels, as well as changes to the levels of nucleotides and key energy metabolism intermediates. PDE6H knockdown induced G1 cell cycle arrest and cell death and reduced mTORC1 signalling in cancer cell lines. Both knockdown and knockout of PDE6H resulted in the suppression of mitochondrial function. HCT116 xenografts revealed that PDE6H deletion, as well as treatment with the PDE5/6 inhibitor sildenafil, slowed down tumour growth and improved survival, while sildenafil treatment did not have an additive effect on slowing the growth of PDE6γ′-deficient tumours.ConclusionsOur results indicate that the changes in cGMP and purine pools, as well as mitochondrial function which is observed upon PDE6γ′ depletion, are independent of the PKG pathway. We show that in HCT116, PDE6H deletion replicates many effects of the dark retina response and identify PDE6H as a new target in preventing cancer cell proliferation and tumour growth.

Biological and computational assessment of new synthesized nicotinamides as potential immunomodulatory VEGFR-2 inhibitors

As an extension to our preceding studies on nicotinamide derivatives as anticancer agents, new nicotinamide-based candidates were designed and synthesized as VEGFR-2 inhibitors. The in vitro cytotoxic activity of the synthesized compounds was evaluated against three human cancer cell lines (MCF-7, HepG-2 and HCT-116). The IC50 values for compound 17 were 2.61± 0.01, 3.20 ± 0.02, and 2.46 ± 0.01 µM, respectively, compared to sorafenib (4.21±0.03, 3.40 ± 0.02, and 5.30 ± 0.04 µM) against MCF-7, HePG-2, and HCT-116. This indicated that compound 17 possess double strength relative to sorafenib against both MCF-7 and HCT-116. Compound 17 was the most promising VEGFR-2 inhibitor with IC50 value of 0.34 μM that was slightly better than that of sorafenib (0.38 μM). Further studies displayed the ability of compound 17 to arrest the growth of HCT-116 cells at the Pre-G1 and S phases. Additionally, compound 17 induced a significant increase in the total apoptosis rate of HCT-116 cells from 1.82 % to 26.69 %. Moreover, it showed high selectivity indices against HCT-116, HepG2, and MCF-7 cancer cells. Furthermore, compound 17 showed potent inhibitory activities on TNF-α and IL-6 and showed a notable rise in caspase-3 level. In addition, the potentiality of the designed derivatives to bind with and inhibit the VEGFR-2 enzyme was indicated by molecular docking assessments. MD simulation studies revealed the stability of compound 17 in the active site of VEGFR-2 for 100 ns. Based on the previous findings, compound 17 appears to be a promising apoptotic VEGFR-2 inhibitor and could potentially direct future efforts towards the development of novel anticancer medications.

Synthesis and molecular docking simulations of novel azepines based on quinazolinone moiety as prospective antimicrobial and antitumor hedgehog signaling inhibitors

A series of novel azepine derivatives based on quinazolinone moiety was synthesized through the reaction of quinazolinone chalcones (2a–d) either with 2-amino aniline in acidic medium to give diazepines (3a–d) or with 2-aminophenol to offer oxazepine (4a–d). The structure of the synthesized compounds was confirmed via melting points, elemental analyses, and different spectroscopic techniques. Moreover, these newly compounds mode of action was investigated in-silico using molecular docking against the outer membrane protein A (OMPA), exo-1,3-beta-glucanase for their antimicrobial activity, and against Smoothened (SMO), transcription factor glioma-associated homology (SUFU/GLI-1), the main proteins of Hedgehog signaling pathway to inspect their anticancer potential. Our results showed that, diazepine (3a) and oxazepine (4a) offered the highest binding energy against the target OMPA/ exo-1,3-beta-glucanase proteins and exhibited the potent antimicrobial activities against E. coli, P. aeruginosa, S. aureus, B. subtilis, C. Albicans and A. flavus. As well, diazepine (3a) and oxazepine (4a) achieved the best results among the other compounds, in their binding energy against the target SMO, SUFU/GLI-1 proteins. The in-vitro cytotoxic study was done for them on panel of cancer cell lines HCT-116, HepG2, and MCF-7 and normal cell line WI-38. Conclusively, it was revealed that molecular docking in-silico simulations and the in-vitro experiments were agreed. As a result, our findings elucidated that diazepine (3a) and oxazepine (4a), have the potential to be used as antimicrobial agents and as possible cancer treatment medications.

Pomegranate extract-loaded sphingosomes for the treatment of cancer: Phytochemical investigations, formulation, and antitumor activity evaluation.

Formulation of Pomegranate Extracts (PE)-loaded sphingosomes as an antitumor therapy for the intravenous and passive targeted delivery to various tumor types, especially that of the breast, colon, and uterus; to increase the therapeutic activity and decrease the adverse effects profile. The pericarp and seeds' juice of Punica granatum were each extracted using D.W. and ethanol. Phytochemical investigation of all extracts was carried out including total phenolics, flavonoids, and anthocyanins contents, the antioxidant activity, as well as HPLC analysis of phenolics and flavonoids. The antitumor potential of all extracts was also tested utilizing three cell lines: MCF-7, HeLa, and HCT116. The candidate extract was chosen for the formulation phase and was entrapped into the sphingosomes using the thin-film hydration method and employing three different PE: lipids weight ratios. The synthesized formulations were characterized for their size, morphological features, zeta potential, entrapment efficiency, and in vitro drug release and kinetics modeling studies. The optimized formula was further analyzed by FTIR spectroscopy and electron microscopy. The antitumor activity of F2 was also investigated using the same cancer cell lines compared to the plant extract. The highest phenolics, flavonoids, and anthocyanins contents were observed in the ethanolic pericarps extract (EPE), followed by the ethanolic seeds extract (ESE). Consequently, EPE showed a higher antitumor activity hence it was selected for the formulation phase. PE-loaded sphingosomes formula (F2) was selected for having the highest EE% (71.64%), and a sustained release profile with the highest in vitro release (42.5±9.44%). By employing the DDSolver, the Weibull model was found the most suitable to describe the PE release kinetics compared to other models. The release mechanism was found to follow Fickian diffusion. Simulated pharmacokinetic parameters have portrayed F2 as the candidate formula, with the highest AUC (536.095) and slowest MDT (0.642 h). In addition, F2 exhibited a significant (p>0.05) stronger and prolonged anticancer effect against MCF-7, HeLa, and HCT116 cell lines at all concentrations tested compared to the free extract. The results proved that sphingosomes are an effective delivery system, improving pharmacological efficacy and reducing serious side effects of anticancer medications and natural products.

Novel N-pyrocatechoyl and N-pyrogalloyl hydrazone antioxidants endowed with cytotoxic and antibacterial activity.

Over the years, pharmacological agents bearing antioxidant merits arose as beneficial in the prophylaxis and treatment of various health conditions. Hazardous effects of radical species hyperproduction disrupt normal cell functioning, thus increasing the possibility for the development of various oxidative stress-associated disorders, such as cancer. Contributing to the efforts for efficient antioxidant drug discovery, a thorough in vitro and in silico assessment of antioxidant properties of 14 newly synthesized N-pyrocatechoyl and N-pyrogalloyl hydrazones (N-PYRs) was accomplished. All compounds exhibited excellent antioxidant potency against the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. The extensive in silico analysis revealed multiple favorable features of N-PYRs to inactivate harmful radical species, which supported the obtained in vitro results. Also, in silico experiments provided insights into the preferable antioxidant pathways. Prompted by these findings, the cytotoxicity effects and the influence on the redox status of cancer HCT-116 cells and healthy fibroblasts MRC-5 were evaluated. These investigations exposed four analogs exhibiting both cytotoxicity and selectivity toward cancer cells. Furthermore, the frequently uncovered antimicrobial potency of hydrazone-type hybrids encouraged investigations on G+ and G- bacterial strains, which revealed the antibacterial potency of several N-PYRs. These findings highlighted the N-PYRs as excellent antioxidant agents endowed with cytotoxic and antibacterial features.

Virtual screening of natural products targeting ubiquitin-specific protease 7

Ubiquitin-specific protease 7 (USP7) is a promising prognostic and druggable target for cancer therapy. Inhibition of USP7 can activate the MDM2-P53 signaling pathway, thereby promoting cancer cell apoptosis. This study based on watvina molecular docking of virtual screening method and biological evaluation found the new USP7 inhibitors targeting catalytic active site. Three hits were screened from 3760 natural products and validated as USP7 inhibitors by enzymatic and kinetic assays. The IC50 values of scutellarein (Scu), semethylzeylastera (DML) and salvianolic acid C (SAC) were 3.017, 6.865 and 8.495 μM, respectively. Further, we reported that the hits could downregulate MDM2 and activate p53 signal pathway in HCT116 cells. Molecular dynamics simulation was used to investigate the binding mechanism of USP7 to Scu, the compound with the best performance, which formed stable contact with Val296, Gln297, Phe409, Tyr465 and Tyr514. These interactions are essential for maintaining the biological activity of Scu. Three natural products are suitable as lead compounds for the development of novel USP7 inhibitors, especially anti-colon cancer drugs. Communicated by Ramaswamy H. Sarma

Controlling cisplatin release by synergistic action of silver-cisplatin on monodispersed spherical silica for targeted anticancer and antibacterial activities

Recently, metal-based nanoformulations have been explored for targeted cancer therapeutics. In this study, synergistic therapeutic action of silver (Ag)-cisplatin nanoparticles loaded on monodispersed spherical silica (MSS) was investigated to evaluate anti-cancer (HCT-116 and HeLa cells) and antibacterial activity (P.aeruginosa, and S.aureus). Four different wt% of Ag (1, 2, 4 and 6 wt%) was loaded on MSS using a wet impregnation method. Characterization using XRD revealed the cubic phase of Ag nanoparticles with (111), (200) and (220) plane on MSS. The chemical state, dispersion of Ag and synergistic cohabitation with cisplatin (Cpt) was identified using diffuse reflectance UV–visible spectroscopy (DR-UV Vis), X-ray photoelectron spectroscopy (XPS), Scanning electron microscopy/energy-dispersive X-ray spectrometry (SEM-EDS) and High-resolution transmission electron microscopy (HR-TEM). Zeta potential analysis indicated an increase in hydrodynamic diameter with high colloidal stability (–23.3 to −28.5 mV). The percentage cumulative Cpt release was studied using automated Franz cell and dialysis membrane technique. The effect of Cpt release on 4 wt%Ag impregnated on different shaped supports was investigated using cubic SBA-16 (Santa Barbara Amorphous), MFI type Ti-ZSM-5 (Zeolite Socony Mobil #5), hexagonal mesosilicalite, nanotube halloysite, and Ag-silicalite, respectively. Drug release study indicates that Ag loading controls the Cpt release. The kinetics of drug release for the different formulations was examined using Korsmeyers-Peppas model. Release constant (k) and release exponent (n) were utilized for the rate of drug release and diffusion mechanisms, respectively. The in vitro study revealed an efficient interaction of nanoformulations promoting both antibacterial and anticancer activities.

Laetiporus sulphureus mushroom extract strongly enhances proapoptotic effect of probiotics Bifidobacterium lactis on HCT-116 cells in a co-culture system

Laetiporus sulphureus is an edible mushroom with significant medicinal potential due to its rich polyphenol composition. Complete activity of polyphenols in the colon depends on the probiotic bacteria which already demonstrated proapoptotic effects on colon cancer cells. Our study uses a simple HoxBan co-culture system in order to approximate real in vivo conditions. Present study examined for the first time the cytotoxic effect of L. sulphureus ethyl acetate extract (EALS) and Bifidobacterium animalis subsp. lactis (BAL) probiotic on colorectal (HCT-116) and healthy (MRC-5) cells. EALS, abundant in polyphenolics detected by HPLC, showed weak cytotoxic activity (MTT assay) on HCT-116 and MRC-5 cells. Strong cytotoxicity of BAL and BAL/EALS on HCT-116 cells (determined by trypan blue staining and flow cytometry) was due to induction of proapoptotic effects. External apoptotic pathway was activated via intensified expression of Caspase 8 and Caspase 3, detected by qPCR method. The results show that EALS strongly enhances the cytotoxic and proapoptotic effects of BAL on HCT-116 cells in the co-culture system. Directions for future research are suggested to ultimately provide the clarification whether the structural change of polyphenols caused by the activity of BAL, or the change in the metabolic activity of BAL by polyphenols, are responsible for exact mechanism of these treatments activity. Also, the consequences of direct contact of BAL and HCT-116 cells should be further examined in appropriate in vivo systems.

Structural Characterization of 5-Substituted Pyrrolo[3,2-d]pyrimidine Antifolate Inhibitors in Complex with Human Serine Hydroxymethyl Transferase 2.

We previously discovered first-in-class multitargeted 5-substituted pyrrolo[3,2-d]pyrimidine antifolates that inhibit serine hydroxymethyltransferase 2 (SHMT2), resulting in potent in vitro and in vivo antitumor efficacies. In this report, we present crystallographic structures for SHMT2 in complex with an expanded series of pyrrolo[3,2-d]pyrimidine compounds with variations in bridge length (3-5 carbons) and the side chain aromatic ring (phenyl, thiophene, fluorine-substituted phenyl, and thiophene). We evaluated structural features of the inhibitor-SHMT2 complexes and correlations to inhibitor potencies (i.e., Kis), highlighting conserved polar contacts and identifying 5-carbon bridge lengths as key determinants of inhibitor potency. Based on the analysis of SHMT2 structural data, we investigated the impact of mutation of Tyr105 in SHMT2 kinetic analysis and studies with HCT116 cells with inducible expression of wild-type and Y105F SHMT2. Increased enzyme inhibition potency by the pyrrolo[3,2-d]pyrimidine inhibitors with Phe105 SHMT2 accompanied an increased growth inhibition of Phe105-expressing HCT116 cells compared to wild-type SHMT2. Pyrrolo[3,2-d]pyrimidine inhibitors with polyglutamate modifications were evaluated for potencies against SHMT2. We determined the crystal structures of SHMT2 in complex with our lead antifolate AGF347 lacking L-glutamate, or as a diglutamate and triglutamate, for comparison with parent AGF347. These data provide the first insights into the influence of antifolate polyglutamylation on SHMT2:inhibitor interactions. Collectively, our results provide new insights into the critical structural determinants of SHMT2 binding by pyrrolo[3,2-d]pyrimidine inhibitors as novel antitumor agents, as well as the first structural characterization of human SHMT2 in complex with polyglutamates of an SHMT2-targeted antifolate.

Elucidation of the mechanism of action of ailanthone in the treatment of colorectal cancer: integration of network pharmacology, bioinformatics analysis and experimental validation.

Background: Ailanthone, a small compound derived from the bark of Ailanthus altissima (Mill.) Swingle, has several anti-tumour properties. However, the activity and mechanism of ailanthone in colorectal cancer (CRC) remain to be investigated. This study aims to comprehensively investigate the mechanism of ailanthone in the treatment of CRC by employing a combination of network pharmacology, bioinformatics analysis, and molecular biological technique. Methods: The druggability of ailanthone was examined, and its targets were identified using relevant databases. The RNA sequencing data of individuals with CRC obtained from the Cancer Genome Atlas (TCGA) database were analyzed. Utilizing the R programming language, an in-depth investigation of differentially expressed genes was carried out, and the potential target of ailanthone for anti-CRC was found. Through the integration of protein-protein interaction (PPI) network analysis, GO and KEGG enrichment studies to search for the key pathway of the action of Ailanthone. Then, by employing molecular docking verification, flow cytometry, Transwell assays, and Immunofluorescence to corroborate these discoveries. Results: Data regarding pharmacokinetic parameters and 137 target genes for ailanthone were obtained. Leveraging The Cancer Genome Atlas database, information regarding 2,551 differentially expressed genes was extracted. Subsequent analyses, encompassing protein-protein interaction network analysis, survival analysis, functional enrichment analysis, and molecular docking verification, revealed the PI3K/AKT signaling pathway as pivotal mediators of ailanthone against CRC. Additionally, the in vitro experiments indicated that ailanthone substantially affects the cell cycle, induces apoptosis in CRC cells (HCT116 and SW620 cells), and impedes the migration and invasion capabilities of these cells. Immunofluorescence staining showed that ailanthone significantly inhibited the phosphorylation of AKT protein and suppressed the activation of the PI3K/AKT signaling pathway, thereby inhibiting the proliferation and metastasis of CRC cells. Conclusion: Therefore, our findings indicate that Ailanthone exerts anti-CRC effects primarily by inhibiting the activation of the PI3K/AKT pathway. Additionally, we propose that Ailanthone holds potential as a therapeutic agent for the treatment of human CRC.

Discovery of anticancer compound possessing potential to bind γ-secretase catalytic subunit and inhibit notch promoter activity

Gamma secretase (GS) is an important therapeutic target in anticancer drug discovery. Increased GS activity activates notch signaling pathway which is associated with cancer stemness and drug resistance in cancer cells. A total of 69,075 natural and their derivative compounds were screened to identify the lead compound on the basis of in silico GS catalytic domain binding potential and in vitro selective anticancer efficacy. STOCK1N-23234 showed higher dock score (−11.82) compared to DAPT (−9.2) in molecular docking experiment and formed hydrogen bond with the key amino acid (Asp385) involve in catalysis process. Molecular dynamics (MD) simulation parameters (RMSD, RMSF, Rg, SASA and hydrogen bond formation) revealed that the STOTCK1N-23234 formed structurally and energetically stable complex with the GS catalytic domain with lower binding energy (−22.79 kcal/mol) compared to DAPT (−16.22 kcal/mol). STOCK1N-23234 showed better toxicity (up to 60%) against colon and breast cancer cells (HCT-116 and MDA-MB-453) at 1–70 µM concentration. Interestingly, STOCK1N-23234 did not showed cytotoxicity against human normal breast cells (MCF-10A). STOCK1N-23234 treatment significantly decreased sphere formation, notch promoter activity, and transcription of notch target genes (Hes-1 and Hey-1) in HCT-116 cells derived colonosphere. Confocal microscopy revealed that STOTCK1N-23234 treatment at test concentration induced apoptosis related morphological changes, reduced mitochondria membrane potential and increased reactive oxygen species production in HCT-116 cells compared to non-treated cells. In conclusion, STOCK1N-23234 is a novel lead natural anticancer compound which requires in depth validation in cancer preclinical models. Communicated by Ramaswamy H. Sarma

GNAS mutation inhibits growth and induces phosphodiesterase 4D expression in colorectal cancer cell lines.

Approximately 5% of colorectal cancers (CRCs) have a gain-of-function mutation in the GNAS gene, which leads to the activation of cAMP-dependent signaling pathways and associates with poor prognosis. We investigated the effect of an activating GNAS mutation in CRC cell lines on gene expression and cell proliferation in vitro, and tumor growth in vivo. GNAS-mutated (GNASmt) HCT116 cells showed stimulated synthesis of cAMP as compared to parental (Par) cells. The most upregulated gene in the GNASmt cells was cAMP-hydrolyzing phosphodiesterase 4D (PDE4D) as detected by RNA sequencing. To further validate our finding, we analyzed PDE4D expression in a set of human CRC tumors (n = 35) and demonstrated overexpression in GNAS mutant CRC tumors as compared to GNAS wild-type tumors. The GNASmt HCT116 cells proliferated more slowly than the Par cells. PDE4 inhibitor Ro 20-1724 and PDE4D subtype selective inhibitor GEBR-7b further suppressed the proliferation of GNASmt cells without an effect on Par cells. The growth inhibitory effect of these inhibitors was also seen in the intrinsically GNAS-mutated SK-CO-1 CRC cell line having high levels of cAMP synthesis and PDE4D expression. In vivo, GNASmt HCT116 cells formed smaller tumors than the Par cells in nude mice. In conclusion, our findings demonstrate that GNAS mutation results in the growth suppression of CRC cells. Moreover, the GNAS mutation-induced overexpression of PDE4D provides a potential avenue to impede the proliferation of CRC cells through the use of PDE4 inhibitors.

Real-time simultaneous refractive index and thickness mapping of sub-cellular biology at the diffraction limit

Mapping the cellular refractive index (RI) is a central task for research involving the composition of microorganisms and the development of models providing automated medical screenings with accuracy beyond 95%. These models require significantly enhancing the state-of-the-art RI mapping capabilities to provide large amounts of accurate RI data at high throughput. Here, we present a machine-learning-based technique that obtains a biological specimen’s real-time RI and thickness maps from a single image acquired with a conventional color camera. This technology leverages a suitably engineered nanostructured membrane that stretches a biological analyte over its surface and absorbs transmitted light, generating complex reflection spectra from each sample point. The technique does not need pre-existing sample knowledge. It achieves 10−4 RI sensitivity and sub-nanometer thickness resolution on diffraction-limited spatial areas. We illustrate practical application by performing sub-cellular segmentation of HCT-116 colorectal cancer cells, obtaining complete three-dimensional reconstruction of the cellular regions with a characteristic length of 30 μm. These results can facilitate the development of real-time label-free technologies for biomedical studies on microscopic multicellular dynamics.

Microbial metabolite trimethylamine-N-oxide induces intestinal carcinogenesis through inhibiting farnesoid X receptor signaling.

Microbial dysbiosis is considered as a hallmark of colorectal cancer (CRC). Trimethylamine-N-oxide (TMAO) as a gut microbiota-dependent metabolite has recently been implicated in CRC development. Nevertheless, evidence relating TMAO to intestinal carcinogenesis remains largely unexplored. Herein, we aimed to examine the crucial role of TMAO in CRC progression. Apcmin/+ mice were treated with TMAO or sterile PBS for 14 weeks. Intestinal tissues were isolated to evaluate the effects of TMAO on the malignant transformation of intestinal adenoma. The gut microbiota of mouse feces was detected by 16S rRNA sequencing analysis. HCT-116 cells were used to provide further evidence of TMAO on the progression of CRC. TMAO administration increased tumor cell and stem cell proliferation, and decreased apoptosis, accompanied by DNA damage and gut barrier impairment. Gut microbiota analysis revealed that TMAO induced changes in the intestinal microbial community structure, manifested as reduced microbiota diversity and beneficial bacteria. Mechanistically, TMAO bound to farnesoid X receptor (FXR), thereby inhibiting the FXR-fibroblast growth factor 15 (FGF15) axis and activating the Wnt/β-catenin signaling pathway, whereas the FXR agonist GW4064 could blunt TMAO-induced Wnt/β-catenin pathway activation. The microbial metabolite TMAO can enhance intestinal carcinogenesis by inhibiting the FXR-FGF15 pathway.

Synthesis of compounds based on the active domain of cabotegravir and their application in inhibiting tumor cells activity.

Structural modification based on existing drugs, which ensures the safety of marketed drugs, is an essential approach in developing new drugs. In this study, we modified the structure of cabotegravir by introducing the front alkyne on the core structure through chemical reaction, resulting in the synthesis of 9 compounds resembling 1,2,3-triazoles. The potential of these new cabotegravir derivatives as tumor suppressors in gastrointestinal tumors was investigated. Based on the MTT experiment, most compounds showed a reduction in the viability of KYSE30 and HCT116 cells. Notably, derivatives 5b and 5h exhibited the most significant inhibitory effects. To further explore the effects of derivatives 5b and 5h on gastrointestinal tumors, KYSE30 cells were chosen as a representative cell line. Both derivatives can effectively curtail the migration and invasion capabilities of KYSE30 cells and induce apoptosis in a dose-dependent manner. We further demonstrated these derivatives induce cell apoptosis in KYSE30 cells by inhibiting the expression of Stat3 protein and Smad2/3 protein. Based on the above results, we suggest they show promise in developing drugs for esophageal squamous cell carcinoma.