Human Colorectal Carcinoma Cells Research Articles

28-Norlupanes and dammaranes from the heartwood of Dipterocarpus costatus Gaertn. f. (Dipterocarpaceae) and their cytotoxic and antiplasmodial activities

Triterpenoids of natural origin are of increasing interest due to their broad spectrum of biological activities. Previous studies on dipterocarpaceous plants have reported that they were valuable source of new active natural compounds. As part of an ongoing search for naturally occurring bioactive triterpenoids from Dipterocarpaceae, phytochemical investigation of Dipterocarpus costatus Gaertn. f. heartwood collected in Thailand was conducted. Seventeen triterpenoids (1-17) belonging to both 28-norlupane and dammarane series were isolated from the corresponding bioactive n-hexane extract. Among them, norlupane 1 was identified as previously undescribed and norlupane 2 was described for the first time from a natural source. Compound structures were elucidated by 1D/2D-NMR spectroscopy and mass (HRMS) spectrometry. All isolated molecules were tested for their cytotoxicity on two human colorectal carcinoma cell lines (i.e. HT-29 and HCT116), as well as for their antiplasmodial activity against chloroquine-resistant strain FcB1 of Plasmodium falciparum. Norlupanes 2 and 4 were shown to exhibit a good cytotoxic activity against HCT 116 (IC50=4.2 µM) and potent antiplasmodial activity (IC50=3.74 µM), respectively.

The two faces of Blastocystis spp.: is it the cause of colorectal cancer (CRC) or a consequence of it?

IntroductionOver the last few years, there has been an increase in the prevalence of <i>Blastocystis</i> spp. in colorectal cancer (CRC) patients. Moreover, various in vitro and in vivo studies have highlighted that intestinal colonisation of <i>Blastocystis</i> spp. has an influence on host immune responses leading to cellular apoptosis and membrane permeability. It has been suggested that <i>Blastocystis</i> spp. is an important risk factor for the worsening of CRC.AimTo present evidence concerning the association between CRC and <i>Blastocystis</i> spp.Material and methodsA review of the literature was performed by searching Science Direct, PubMed, Scopus and Google Scholar databases up to December, 2023.Results and discussionOut of all in vitro and in vivo studies selected for this review, the majority of them have confirmed a significantly higher prevalence of <i>Blastocystis</i> spp. in colorectal cancer patients in comparison to the control groups. Several in vitro human colorectal carcinoma cell line studies have shown significant cytopathic and immunological effects of <i>Blastocystis</i> spp. Additionally, in vivo experimental animal model studies have shown that <i>Blastocystis</i> spp. infection significantly contributed to large intestinal polyp (colorectal adenoma) formation and the progression of colorectal carcinogenesis.ConclusionsThese studies strongly support suggestions that <i>Blastocystis</i> spp. could be an important factor to existing CRC development by influencing the host immune response and increasing oxidative damage.

Irciniaplysins A-D: New Psammaplysin Derivatives from Philippine Marine Sponge Ircinia sp.

Four new psammaplysin derivatives (1-4) with fatty acyl substituents, designated irciniaplysins A-D, and three known psammaplysins (5-7) were isolated from a marine sponge Ircinia sp. Their structures were elucidated using extensive spectroscopic analyses. The positions of the double bonds and the branch points of the fatty acyl side chains were determined by GC-MS analysis of their fatty acid methyl ester (FAME) derivatives. Irciniaplysins A (1) and B (2) contained an unusual long-chain fatty acyl substituent with a 5,9-diene unit. The isolated compounds were evaluated for their cytotoxic activity against the human colorectal carcinoma (HCT 116) cells, however, none of these compounds showed significant activity.

Inhibition of Human Colorectal Cancer by a Natural Product 7-Acetylhorminone and Interactions with BSA/HSA: Multispectral Analysis and In Silico and In Vitro Studies.

We have semi-synthesized a natural product 7-acetylhorminone from crude extract of Premna obtusifolia (Indian headache tree), which is active against colorectal cancer after probation through computational screening methods as it passed through the set parameters of pharmacokinetics (most important nonblood-brain barrier permeant) and drug likeliness (e.g., Lipinski's, Ghose's, Veber's rule) which most other phytoconstituents failed to pass combined with docking with EGFR protein which is highly upregulated in the colorectal carcinoma cell. The structure of 7-acetylhorminone was confirmed by single crystal X-ray diffraction studies and 1H NMR, 13C NMR, and COSY studies. To validate the theoretical studies, first, in vitro experiments were carried out against human colorectal carcinoma cell lines (HCT116) which revealed the potent cytotoxic efficacy of 7-acetylhorminone and verified preliminary investigation. Second, the drugability of 7-acetylhorminone interaction with serum albumin proteins (HSA and BSA) is evaluated both theoretically and experimentally via steady-state fluorescence spectroscopic studies, circular dichroism, isothermal titration calorimetry, and molecular docking. In summary, this study reveals the applicability of 7-acetylhorminone as a potent drug candidate or as a combinatorial drug against colorectal cancer.

Structure-Activity Relationship Study of Biselyngbyolide B Reveals Mitochondrial Fission-Induced Cytotoxicity in Cancer.

A systematic structure-activity relationship study of the potent anticancer marine macrolide biselyngbyolide B has been accomplished. A total of 11 structural variants of the parent natural product, of which 2 are natural analogues, have been studied against a human colorectal carcinoma cell line. The requisite functional units of the parent molecule responsible for the cytotoxic activities have been disclosed. Biselyngbyolide C, one of the natural analogues of biselyngbyolide B, has been studied in depth to explore its molecular mechanism. Interestingly, the in vitro data demonstrated an induction of dynamin-related protein 1-mediated mitochondrial fission and reactive oxygen species production which led to activation of ASK1/P38/JNK-mediated apoptosis in colon cancer cells as an important pathway for biselyngbyolide B-mediated cytotoxicity. Notably, this study revealed that a macrolide participated in mitochondrial fission to promote apoptosis of cancer cells, providing new insight.

Anti-angiogenic and anti-tumour activities of Lignosus rhinocerus (Cooke) Ryvarden water extracts on HCT116 human colorectal carcinoma cells implanted in chick embryos

Ethnopharmacological relevanceThe sclerotium of Lignosus rhinocerus (Cooke) Ryvarden is used by the local communities in Southeast Asia and China to treat cancer, asthma, fever, and other ailments based on traditional knowledge. The sclerotial water extracts were previously reported to exhibit cytotoxic, apoptotic, and immunomodulatory activities – providing a scientific basis for its use in treating cancer; however, there is still a lack of evidence on its potential anti-angiogenic activity. Aim of the studyThis study aimed to investigate the toxicity, anti-angiogenic, and anti-tumour activities of the hot-water and cold-water extracts of L. rhinocerus using HCT116 human colorectal carcinoma cells implanted in the chick chorioallantoic membrane (CAM) model. Materials and methodsThe toxicity of L. rhinocerus extracts towards the chick embryos was determined 24 h post-treatment. The anti-angiogenic activity of the extracts was then investigated at 0.1–10 μg/embryo (6.7–670 μg/mL) at targeted blood vessels. The anti-tumour effect of selected extracts against the HCT116 human colorectal carcinoma cells xenografted onto the chick embryos was also studied. ResultsThe cold-water extracts of L. rhinocerus displayed strong in ovo toxicity (LC50: 1.2–37.7 μg/mL) while the hot-water extracts are non-toxic up to 670 μg/mL. Among the extracts, the hot-water extracts demonstrated the highest anti-angiogenic activity with 44.0 ± 17.7% reduction of capillary diameter (relative to the saline-treated control). Moreover, treatment of the HCT116 cells xenografted onto the chick embryos with the hot-water extracts resulted in smaller tumour size and lower number of blood vessels compared to the saline-treated control. ConclusionsThe hot-water extracts of L. rhinocerus sclerotium demonstrated anti-angiogenic and anti-tumour activities but most of the cold-water extracts at similar concentrations were devoid of that. Our findings provide further scientific validation of the medicinal use of the sclerotium in treating cancer and thus, expanding our knowledge on the possible mechanism of its anti-cancer effect apart from direct cytotoxicity, induction of apoptosis and immunomodulation that have been studied thus far.

Proteasome activation is critical for cell death induced by inhibitors of polo-like kinase 1 (PLK1) in multiple cancers

Inhibitors of polo-like kinase (PLK) are currently being evaluated as anticancer drugs. However, the molecular mechanism of PLK inhibitor-induced cell death is not fully understood. In this study, we found that GW843682X and BI2536, two inhibitors of PLK1, significantly induced cell death in multiple type cells. The induction of cell death was related to the preferring expression of PLK1. However, in human umbilical vascular endothelial cells (HUVEC) and human colorectal carcinoma cells, which expressed higher levels of both PLK1 and PLK2, PLK1 inhibitors induced very low levels of cell death. Clinical analysis reveals PLK1 presence in 26 of 30 NPC tumor tissues. In in vivo NPC lung metastasis nude mouse models, PLK1 inhibitors decreased NPC progress. Mechanistically, the PLK1 inhibitor did not activate p53, and the cell death was not reversed by p53 inhibition. Moreover, PLK1 inhibitor-induced cell death was PARP- and caspase-independent. Although PLK1 inhibitors induced down-regulation of calpain inhibitor calpastatin and calpain was activated by PLK1 inhibition, calpain blocking did not reverse cell death induced by PLK1 inhibitors, suggesting the non-involvement of calpain. Surprisingly, we found that PLK1 inhibitors induced the activation of proteasome, and the treatment of cells with PLK1 inhibitors reduced the levels of ubiquitinated proteins. And proteasome inhibitors reversed cell death induced by PLK1 inhibitors in various cell types in which PLK1 was preferentially expressed. Moreover, PLK1 inhibition reversed the degradation of proteins including p53, caspase 8, PARP and calpastatin. These results suggest that the activation of proteasome is critical for cell death induced by PLK1 inhibition.

Abstract LB168: A selective, potent, and orally bioavailable CDK7 inhibitor demonstrates superior anti-cancer activity in colorectal cancer models

Abstract Dysregulated cell cycle and gene expression are pivotal factors in cancer development and the regulators of these processes are attractive targets for cancer treatment. The success of CDK4/6 inhibitors such as palbociclib, ribociclib, and abemaciclib underscores the promising role of targeting cell cycle regulators in anti-cancer therapy. On the other hand, a robust correlation has been established between the overexpression of oncogenes and super-enhancer (SE). The genes associated with SE show increased vulnerability to transcription factor disruption compared to genes with typical enhancers. It has been demonstrated in previous studies that inhibiting transcription factors can effectively decrease the expression of SE-associated oncogenes, including c-MYC, thereby highlighting potential anticancer effects for oncogene-addicted cancers. Currently, agents targeting transcription factors are undergoing clinical trials, offering promising therapeutic options for patients with various types of cancers. CDK7 plays a crucial role in orchestrating cell cycle progression and transcription by phosphorylating CDKs, the C-terminal domain of RNA polymerase II, and hormone receptors like estrogen and androgen receptors. The inhibition of CDK7 has been shown to effectively inhibit tumor growth through various mechanisms, including inducing cell cycle arrest and inhibiting transcription, thereby leading to the death of cancer cells. Notably, ongoing clinical studies have demonstrated that small-molecule CDK7 inhibitors effectively control cancer progression as a stand-alone and in combination with other anti-cancer agents. C-16661, a selective, potent, orally bioavailable, and reversible ATP-competitive inhibitor of CDK7, displayed a single-digit nanomolar IC50 value in both biochemical and cellular assays. It exhibited excellent selectivity in a kinase panel assay, and remarkable potency in a human cancer cell line panel assay. C-16661 treatment resulted in strong inhibition of both its direct and indirect downstream targets including pS5-POLII, pT161-CDK1, c-MYC, and MCL1 in HCT116, a human colorectal carcinoma cell line. While C-16661 exhibited good PK profiles in animals, it demonstrated excellent tumor growth inhibition without any tolerability issues in a colorectal cancer CDX mouse efficacy model. Citation Format: Heuijoon Park, Kyoungwan Seo, Jihyun Yu, Jieun Kim, Min Sung Joo, Sungeun Kim, Joonhyung Lee, Jeonghyeon Lee, Eun-Jung Kim, Joonwoo Nam, Wooseok Han. A selective, potent, and orally bioavailable CDK7 inhibitor demonstrates superior anti-cancer activity in colorectal cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB168.

Evaluation of Antiproliferative and Cytotoxic potential of organic solvent fractions of potential plant sources against Human colorectal carcinoma cell lines

People have been relying and using various medicinal sources for ages throughout the world for alleviation of different ailments. Eighty percent of people worldwide depend on herbal medication for their fundamental medical needs, according to a World Health Organization (WHO) survey. The need for plant-based medications has led to increased studies into the extraction of therapeutic properties from plants. Bioactive compounds are secondary metabolites, sometimes termed nutraceuticals, present in food, providing health benefits.

The solute carrier SLC7A1 may act as a protein transporter at the blood-brain barrier

Despite extensive research, targeted delivery of substances to the brain still poses a great challenge due to the selectivity of the blood-brain barrier (BBB). Most molecules require either carrier- or receptor-mediated transport systems to reach the central nervous system (CNS). These transport systems form attractive routes for the delivery of therapeutics into the CNS, yet the number of known brain endothelium-enriched receptors allowing the transport of large molecules into the brain is scarce. Therefore, to identify novel BBB targets, we combined transcriptomic analysis of human and murine brain endothelium and performed a complex screening of BBB-enriched genes according to established selection criteria. As a result, we propose the high-affinity cationic amino acid transporter 1 (SLC7A1) as a novel candidate for transport of large molecules across the BBB. Using RNA sequencing and in situ hybridization assays, we demonstrated elevated SLC7A1 gene expression in both human and mouse brain endothelium. Moreover, we confirmed SLC7A1 protein expression in brain vasculature of both young and aged mice. To assess the potential of SLC7A1 as a transporter for larger proteins, we performed internalization and transcytosis studies using a radiolabelled or fluorophore-labelled anti-SLC7A1 antibody. Our results showed that SLC7A1 internalised a SLC7A1-specific antibody in human colorectal carcinoma (HCT116) cells. Moreover, transcytosis studies in both immortalised human brain endothelial (hCMEC/D3) cells and primary mouse brain endothelial cells clearly demonstrated that SLC7A1 effectively transported the SLC7A1-specific antibody from luminal to abluminal side. Therefore, here in this study, we present for the first time the SLC7A1 as a novel candidate for transport of larger molecules across the BBB.

Structural basis for the preservation of a subset of topologically associating domains in interphase chromosomes upon cohesin depletion

Compartment formation in interphase chromosomes is a result of spatial segregation between euchromatin and heterochromatin on a few megabase pairs (Mbp) scale. On the sub-Mbp scales, topologically associating domains (TADs) appear as interacting domains along the diagonal in the ensemble averaged Hi-C contact map. Hi-C experiments showed that most of the TADs vanish upon deleting cohesin, while the compartment structure is maintained, and perhaps even enhanced. However, closer inspection of the data reveals that a non-negligible fraction of TADs is preserved (P-TADs) after cohesin loss. Imaging experiments show that, at the single-cell level, TAD-like structures are present even without cohesin. To provide a structural basis for these findings, we first used polymer simulations to show that certain TADs with epigenetic switches across their boundaries survive after depletion of loops. More importantly, the three-dimensional structures show that many of the P-TADs have sharp physical boundaries. Informed by the simulations, we analyzed the Hi-C maps (with and without cohesin) in mouse liver and human colorectal carcinoma cell lines, which affirmed that epigenetic switches and physical boundaries (calculated using the predicted 3D structures using the data-driven HIPPS method that uses Hi-C as the input) explain the origin of the P-TADs. Single-cell structures display TAD-like features in the absence of cohesin that are remarkably similar to the findings in imaging experiments. Some P-TADs, with physical boundaries, are relevant to the retention of enhancer–promoter/promoter–promoter interactions. Overall, our study shows that preservation of a subset of TADs upon removing cohesin is a robust phenomenon that is valid across multiple cell lines.

Structural basis for the preservation of a subset of topologically associating domains in interphase chromosomes upon cohesin depletion.

Compartment formation in interphase chromosomes is a result of spatial segregation between euchromatin and heterochromatin on a few megabase pairs (Mbp) scale. On the sub-Mbp scales, topologically associating domains (TADs) appear as interacting domains along the diagonal in the ensemble averaged Hi-C contact map. Hi-C experiments showed that most of the TADs vanish upon deleting cohesin, while the compartment structure is maintained, and perhaps even enhanced. However, closer inspection of the data reveals that a non-negligible fraction of TADs is preserved (P-TADs) after cohesin loss. Imaging experiments show that, at the single-cell level, TAD-like structures are present even without cohesin. To provide a structural basis for these findings, we first used polymer simulations to show that certain TADs with epigenetic switches across their boundaries survive after depletion of loops. More importantly, the three-dimensional structures show that many of the P-TADs have sharp physical boundaries. Informed by the simulations, we analyzed the Hi-C maps (with and without cohesin) in mouse liver and human colorectal carcinoma cell lines, which affirmed that epigenetic switches and physical boundaries (calculated using the predicted 3D structures using the data-driven HIPPS method that uses Hi-C as the input) explain the origin of the P-TADs. Single-cell structures display TAD-like features in the absence of cohesin that are remarkably similar to the findings in imaging experiments. Some P-TADs, with physical boundaries, are relevant to the retention of enhancer-promoter/promoter-promoter interactions. Overall, our study shows that preservation of a subset of TADs upon removing cohesin is a robust phenomenon that is valid across multiple cell lines.

Rosemary essential oil potentiates the antitumour activity of 5-fluorouracil in human colorectal carcinoma cells.

Improving response rates in colorectal cancer (CRC) is an urgent clinical need. This study aimed to explore the synergistic action of Lebanese rosemary essential oil (REO) and 5-fluorouracil (5-FU) in HCT116 CRC cells. We tested the cell viability of monotherapy and combination therapy. The combination index was calculated using CompuSyn software to evaluate drug-drug interactions and the level of synergistic cytotoxicity. We also evaluated cell migration and cytopathology. Furthermore, cell apoptosis-related proteins (i.e. Bax and Bcl-2) were measured by Western blot analysis. The REO/5-FU combination synergistically reduced cell viability, effectively decreased cell migration, and increased the Bax/Bcl-2 ratio in HCT116 cells. This triggered a proapoptotic morphology and initiated an apoptotic cascade in HCT116 cells, as indicated by a higher Bax/Bcl-2 ratio. Our results provide evidence of the REO/5-FU combination as a better approach to improve 5-FU anticancer efficacy and allow the use of lower 5-FU doses due to the adjuvant effect of REO.

Unveiling biological activities of biosynthesized starch/silver-selenium nanocomposite using Cladosporium cladosporioides CBS 174.62

Background and objectivesMicrobial cells capability to tolerate the effect of various antimicrobial classes represent a major worldwide health concern. The flexible and multi-components nanocomposites have enhanced physicochemical characters with several improved properties. Thus, different biological activities of biosynthesized starch/silver-selenium nanocomposite (St/Ag-Se NC) were assessed.MethodologyThe St/Ag-Se NC was biosynthesized using Cladosporium cladosporioides CBS 174.62 (C. cladosporioides) strain. The shape and average particle size were investigated using scanning electron microscope (SEM) and high-resolution transmission electron microscope (HR-TEM), respectively. On the other hand, the St/Ag-Se NC effect on two cancer cell lines and red blood cells (RBCs) was evaluated and its hydrogen peroxide (H2O2) scavenging effect was assessed. Moreover, its effects on various microbial species in both planktonic and biofilm growth forms were examined.ResultsThe St/Ag-Se NC was successfully biosynthesized with oval and spherical shape and a mean particle diameter of 67.87 nm as confirmed by the HR-TEM analysis. St/Ag-Se NC showed promising anticancer activity toward human colorectal carcinoma (HCT-116) and human breast cancer (MCF-7) cell lines where IC50 were 21.37 and 19.98 µg/ml, respectively. Similarly, little effect on RBCs was observed with low nanocomposite concentration. As well, the highest nanocomposite H2O2 scavenging activity (42.84%) was recorded at a concentration of 2 mg/ml. Additionally, Staphylococcus epidermidis (S. epidermidis) ATCC 12,228 and Candida albicans (C. albicans) ATCC 10,231 were the highly affected bacterial and fungal strains with minimum inhibitory concentrations (MICs) of 18.75 and 50 µg/ml, respectively. Moreover, the noticeable effect of St/Ag-Se NC on microbial biofilm was concentration dependent. A high biofilm suppression percentage, 87.5% and 68.05%, were recorded with S. epidermidis and Staphylococcus aureus (S. aureus) when exposed to 1 mg/ml and 0.5 mg/ml, respectively.ConclusionThe biosynthesized St/Ag-Se NC showed excellent antioxidant activity, haemocompatibility, and anti-proliferative effect at low concentrations. Also, it exhibited promising antimicrobial and antibiofilm activities.

Retraction Note: Syndecan-1 suppresses cell growth and migration via blocking JAK1/STAT3 and Ras/Raf/MEK/ERK pathways in human colorectal carcinoma cells

Retraction Note: Syndecan-1 suppresses cell growth and migration via blocking JAK1/STAT3 and Ras/Raf/MEK/ERK pathways in human colorectal carcinoma cells

The effects of meloxicam, lornoxicam, ketoprofen, and dexketoprofen on human cervical, colorectal, and mammary carcinoma cell lines

Targeting the inflammation-related molecules with nonsteroidal anti-inflammatory drugs (NSAIDs) represents a promising approach for cancer prevention/therapy. We evaluated the in vitro anticancer effects of meloxicam, lornoxicam, ketoprofen, and dexketoprofen on the proliferation, migration, and apoptosis of human cervical, colorectal, and mammary carcinoma cells. The antiproliferative activity and cytotoxicity of tested NSAIDs on HeLa, HT-29, and MCF-7 cell lines were assessed by the MTT test. The apoptosis-inducing potential was analyzed by fluorescent staining with acridine orange/ethidium bromide and DAPI. Migration activity was assessed by a wound-healing scratch assay. The tested NSAIDs reduced the viability of the used tumor cell lines. The cytomorphological analysis revealed reduced cell density and mitotic activity and the presence of cells with morphological features of early and late apoptosis. Significant inhibition of the migration capacity was established as well. In conclusion, NSAIDs could be candidates for the development of new pharmacological strategies for the treatment and prevention of cancer.

Targeting mitochondrial bioenergetics by combination treatment with imatinib and dichloroacetate in human erythroleukemic K‑562 and colorectal HCT‑116 cancer cells.

Tumor malignant cells are characterized by dysregulation of mitochondrial bioenergetics due to the 'Warburg effect'. In the present study, this metabolic imbalance was explored as a potential target for novel cancer chemotherapy. Imatinib (IM) downregulates the expression levels of SCΟ2 and FRATAXIN (FXN) genes involved in the heme‑dependent cytochrome c oxidase biosynthesis and assembly pathway in human erythroleukemic IM‑sensitive K‑562 chronic myeloid leukemia cells (K‑562). In the present study, it was investigated whether the treatment of cancer cells with IM (an inhibitor of oxidative phosphorylation) separately, or together with dichloroacetate (DCA) (an inhibitor of glycolysis), can inhibit cell proliferation or cause death. Human K‑562 and IM‑chemoresistant K‑562 chronic myeloid leukemia cells (K‑562R), as well as human colorectal carcinoma cells HCT‑116 (+/+p53) and (‑/‑p53, with double TP53 knock-in disruptions), were employed. Treatments of these cells with either IM (1 or 2µM) and/or DCA (4mΜ) were also assessed for the levels of several process biomarkers including SCO2, FXN, lactate dehydrogenase A, glyceraldehyde‑3‑phosphate dehydrogenase, pyruvate kinase M2, hypoxia inducing factor‑1a, heme oxygenase‑1, NF‑κB, stem cell factor and vascular endothelial growth factor via western blot analysis. Computational network biology models were also applied to reveal the connections between the ten proteins examined. Combination treatment of IM with DCA caused extensive cell death (>75%) in K‑562 and considerable (>45%) in HCT‑116 (+/+p53) cultures, but less in K‑562R and HCT‑116 (‑/‑p53), with the latter deficient in full length p53 protein. Such treatment, markedly reduced reactive oxygen species levels, as measured by flow‑cytometry, in K‑562 cells and affected the oxidative phosphorylation and glycolytic biomarkers in all lines examined. These findings indicated, that targeting of cancer mitochondrial bioenergetics with such a combination treatment was very effective, although chemoresistance to IM in leukemia and the absence of a full length p53 in colorectal cells affected its impact.

Synthesis, characterization, and application of honey stabilized inulin nanoparticles as colon targeting drug delivery carrier

Inulin (INU) is a versatile natural polysaccharide primarily derived from chicory roots. INU possesses the unique quality of evading digestion or fermentation in the early stages of the human digestive tract, instead reaching the lower colon directly. Exploiting on this distinctive attribute, INU finds application in the creation of targeted carrier systems for delivering drugs tailored to colon-related diseases. This study presents a novel method for synthesizing highly stable and non-aggregatory inulin nanoparticles (INU NPs) by ionotropic gelation method, using calcium chloride as crosslinker and natural honey as a stabilizing agent. Different formulation and process parameters were optimized for the synthesis of monodispersed INU NPs. These INU NPs efficiently encapsulated a hydrophilic drug irinotecan hydrochloride trihydrate (IHT) and drug loaded formulation (IINPs) demonstrated excellent colloidal and storage stabilities. Notably, these IINPs exhibited pH-dependent drug release, suggesting potential for colon-specific drug delivery. Anticancer activity of the NPs was found significantly higher in comparison to IHT through cytotoxicity and apoptosis studies against human colorectal carcinoma cells. Overall, this study revealed that the INU NPs synthesized by ionotropic gelation will be an efficient nanocarrier system for colon-targeted drug delivery due to their exceptional biocompatibility and stability in stomach and upper intestinal conditions.

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

Antiproliferative and Antitelomerase Effects of Silymarin on Human Colorectal and Hepatocellular Carcinoma Cells.

Silymarin, a widely-used hepatoprotective agent, has shown antitumor properties in both in vitro and animal studies. Currently, there is limited knowledge regarding silymarin's antitelomerase effects on human colorectal cancer and hepatocyte carcinoma cells. In this study, we investigated the antiproliferative and antitelomerase effects of silymarin on four human colorectal cancer and HepG2 hepatocyte carcinoma cell lines. The cell viability and telomerase activity were assessed using MTT and the telomerase repeat amplification protocol assay, respectively. We also investigated the effects of silymarin on the expression of human telomerase reverse transcriptase and its promoter methylation in HepG2 cells by real-time RT-PCR and methylation-specific PCR, respectively. Silymarin treatment inhibited cell proliferation and telomerase activity in all cancer cells. After 24 h of treatment, silymarin exhibited IC50 values ranging from 19 - 56.3 µg/mL against these cancer cells. A 30-min treatment with silymarin at the IC50 concentration effectively inhibited telomerase activity in cell-free extracts of both colorectal cancer and hepatocyte carcinoma cells. Treatment of HepG2 cells with 10 and 30 µg/mL of silymarin for 48 h resulted in a decrease in human telomerase reverse transcriptase expression to 75 and 35% of the level observed in the untreated control (p < 0.01), respectively. Treatment with silymarin (10, 30, and 60 µg/mL) for 48 h did not affect human telomerase reverse transcriptase promoter methylation in HepG2 cells. In conclusion, our findings suggest that silymarin inhibits cancer cell growth by directly inhibiting telomerase activity and downregulating its human telomerase reverse transcriptase catalytic subunit. However, silymarin did not affect human telomerase reverse transcriptase promoter methylation at the concentrations of 10 - 60 µg/mL used in this study.