Celecoxib-Induced Modulation of Colon Cancer CD133 Expression Occurs through AKT Inhibition and Is Monitored by 89Zr Immuno-PET

We developed an immuno-PET technique that monitors modulation of tumor CD133 expression, which is required for the success of CD133-targeted therapies. Methods. Anti-CD133 antibodies were subjected to sulfhydryl moiety-specific 89Zr conjugation. 89Zr-CD133 IgG was evaluated for specific activity and radiolabel stability. Colon cancer cells underwent binding assays and Western blotting. Biodistribution and PET studies were performed in mice. Results. 89Zr-CD133 IgG showed excellent target specificity with 97.2 ± 0.7 % blocking of HT29 cell binding by an excess antibody. Intravenous 89Zr-CD133 IgG followed biexponential blood clearance and showed CD133-specific uptake in HT29 tumors. 89Zr-CD133 IgG PET/CT and biodistribution studies confirmed high HT29 tumor uptake with lower activities in the blood and normal organs. In HT29 cells, celecoxib dose-dependently decreased CD133 expression and 89Zr-CD133 IgG binding that reached 19.9 ± 2.1 % ( P < 0.005 ) and 50.3 ± 10.9 % ( P < 0.001 ) of baseline levels by 50 μM, respectively. Celecoxib treatment of mice significantly suppressed tumor CD133 expression to 67.5 ± 7.8 % of controls ( P < 0.005 ) and reduced tumor 89Zr-CD133 IgG uptake from 15.5 ± 1.4 % at baseline to 12.3 ± 2.0 % ID / g ( P < 0.01 ). Celecoxib-induced CD133 reduction in HT29 cells and tumors was associated with substantial suppression of AKT activation. There were also reduced HIF-1α accumulation and IκBα/NFκB phosphorylation. Conclusion. 89Zr-CD133 IgG PET provides high-contrast tumor imaging and monitors celecoxib treatment-induced modulation of tumor CD133 expression, which was found to occur through AKT inhibition. This technique may thus be useful for screening drugs that can effectively suppress colon cancer stem cells.

A GdW10@PDA-CAT Sensitizer with High-Z Effect and Self-Supplied Oxygen for Hypoxic-Tumor Radiotherapy

Anticancer treatment is largely affected by the hypoxic tumor microenvironment (TME), which causes the resistance of the tumor to radiotherapy. Combining radiosensitizer compounds and O2 self-enriched moieties is an emerging strategy in hypoxic-tumor treatments. Herein, we engineered GdW10@PDA-CAT (K3Na4H2GdW10O36·2H2O, GdW10, polydopamine, PDA, catalase, CAT) composites as a radiosensitizer for the TME-manipulated enhancement of radiotherapy. In the composites, Gd (Z = 64) and W (Z = 74), as the high Z elements, make X-ray gather in tumor cells, thereby enhancing DNA damage induced by radiation. CAT can convert H2O2 to O2 and H2O to enhance the X-ray effect under hypoxic TME. CAT and PDA modification enhances the biocompatibility of the composites. Our results showed that GdW10@PDA-CAT composites increased the efficiency of radiotherapy in HT29 cells in culture. This polyoxometalates and O2 self-supplement composites provide a promising radiosensitizer for the radiotherapy field.

An Optical Tweezers-Based Single-Cell Manipulation and Detection Platform for Probing Real-Time Cancer Cell Chemotaxis and Response to Tyrosine Kinase Inhibitor PD153035

We presented an approach to address cancer cell chemotaxis and response to tyrosine kinase inhibitor PD153035 at the single-cell level. We applied an optical tweezer system together with the platform at the single-cell level to manipulate an epidermal growth factor (EGF)-coated bead positioned close to the filopodia to locally stimulate HT29 cells, the human colon cancer cell line overexpressing the EGF receptor (EGFR). To address cancer cell chemotaxis, a single-cell movement model was also proposed to quantify the propagation speed at the leading and trailing edges of the cell along the chemosensing axis. This study focused on three perspectives: probing the chemosensing process mediated by EGF/EGFR signaling, investigating the mode of locomotion during the EGF-coated bead stimulation, and quantifying the effect of PD153035 on the EGF–EGFR transport pathway. The results showed that the filopodial actin filament is a sensory system for EGF detection. In addition, HT29 cells may use the filopodial actin filament to distinguish the presence or absence of the chemoattractant EGF. Furthermore, we demonstrated the high selectivity of PD153035 for EGFR and the reversibility of binding to EGFR. We anticipate that the proposed single-cell method could be applied to construct a rapid screening method for the detection and therapeutic evaluation of many types of cancer during chemotaxis.

Camptothecin Encapsulated in β-Cyclodextrin-EDTA-Fe3O4 Nanoparticles Induce Metabolic Reprogramming Repair in HT29 Cancer Cells through Epigenetic Modulation: A Bioinformatics Approach

Cancer progresses through a distinctive reprogramming of metabolic pathways directed by genetic and epigenetic modifications. The hardwired changes induced by genetic mutations are resilient, while epigenetic modifications are softwired and more vulnerable to therapeutic intervention. Colon cancer is no different. This gives us the need to explore the mechanism as an attractive therapeutic target to combat colon cancer cells. We have previously established the enhanced therapeutic efficacy of a newly formulated camptothecin encapsulated in β-cyclodextrin-EDTA-Fe3O4 nanoparticles (CPT-CEF) in colon cancer cells. We furthered this study by carrying out RNA sequencing (RNA-seq) to underscore specific regulatory signatures in the CPT-CEF treated versus untreated HT29 cells. In the study, we identified 95 upregulated and 146 downregulated genes spanning cellular components and molecular and metabolic functions. We carried out extensive bioinformatics analysis to harness genes potentially involved in epigenetic modulation as either the cause or effect of metabolic rewiring exerted by CPT-CEF. Significant downregulation of 13 genes involved in the epigenetic modulation and 40 genes from core metabolism was identified. Three genes, namely, DNMT-1, POLE3, and PKM-2, were identified as the regulatory overlap between epigenetic drivers and metabolic reprogramming in HT29 cells. Based on our results, we propose a possible mechanism that intercepts the two functional axes, namely epigenetic control, and metabolic modulation via CPT-CEF in colon cancer cells, which could skew cancer-induced metabolic deregulation towards metabolic repair. Thus, the study provides avenues for further validation of transcriptomic changes affected by these deregulated genes at epigenetic level, and ultimately may be harnessed as targets for regenerating normal metabolism in colon cancer with better treatment potential, thereby providing new avenues for colon cancer therapy.

Profiling Activins and Follistatin in Colorectal Cancer According to Clinical Stage, Tumour Sidedness and Smad4 Status

This study explored the roles of activins and follistatin in colorectal cancers. Paired malignant and normal colonic tissues were collected from archived paraffin-embedded (n = 90 patients) alongside fresh (n = 40 patients) specimen cohorts. Activin β-subunits, follistatin and Smad4 mRNAs and proteins were measured by real-time PCR and immunohistochemistry (IHC). Mature activin-A, -B, -AB and follistatin proteins were measured by ELISA. Cancer tissues having ≤ the 20th percentile of the Smad4 IHC score were considered as low (L-S4) group. The Smad4-intact SW480 and Smad4-null HT29 colon cancer cell lines were treated with activins and follistatin, and cell cycle was analysed by flow cytometry. The cell cycle inducing (CCND1/CCND3) and inhibitory (p21/p27) proteins alongside the survival (survivin/BCL2) and pro-apoptosis (Casp-8/Casp-3) markers were measured by immunofluorescence. Thirty-nine patients had right-sided cancers (30%) and showed higher rates of L-S4 tumours (n = 17; 13.1%) alongside worse clinicopathological characteristics relative to left-sided cancers. The βA-subunit and activin-A increased, whilst βB-subunit and activin-AB decreased, in malignant sites and the late-stage cancers revealed the greatest abnormalities. Interestingly, follistatin declined markedly in early-stage malignant tissues, whilst increased significantly in the advanced stages. All activin molecules were comparable between the early stage right- and left-sided tumours, whereas the late-stage right-sided cancers and L-S4 tumours showed more profound deregulations. In vitro, activin-A increased the numbers of the SW480 cells in sub-G1 and G0/G1-phases, whereas reduced the HT29 cell numbers in the sub-G1 phase with simultaneous increases in the G0/G1 and S phases. The p21/p27/Casp-8/Casp-3 proteins escalated, whilst CCND1/CCND3/BCL2/survivin declined in the SW480 cells following activin-A, whereas activin-A only promoted p21 and p27 alongside reduced CCND3 in the HT29 cells. By contrast, activin-AB increased the numbers of SW480 and HT29 cells in Sub-G1 and G0/G1-phases and promoted the anti-cancer and reduced the oncogenic proteins in both cell lines. In conclusion, activins and follistatin displayed stage-dependent dysregulations and were markedly altered during the advanced stages of right-sided and L-S4 cancers. Moreover, the activin-A actions in CRC could be Smad4-dependent, whereas activin-AB may act as a Smad4-independent tumour suppressor protein.

Topoisomerase poisoning by the flavonoid nevadensin triggers DNA damage and apoptosis in human colon carcinoma HT29 cells

Nevadensin, an abundant polyphenol of basil, is reported to reduce alkenylbenzene DNA adduct formation. Furthermore, it has a wide spectrum of further pharmacological properties. The presented study focuses the impact of nevadensin on topoisomerases (TOPO) in vitro. Considering the DNA-intercalating properties of flavonoids, first, minor groove binding properties (IC50 = 31.63 µM), as well as DNA intercalation (IC50 = 296.91 µM) of nevadensin, was found. To determine potential in vitro effects on TOPO I and TOPO IIα, the relaxation and decatenation assay was performed in a concentration range of 1–500 µM nevadensin. A partial inhibition was detected for TOPO I at concentrations ≥ 100 µM, whereas TOPO IIα activity is only inhibited at concentrations ≥ 250 µM. To clarify the mode of action, the isolating in vivo complex of enzyme assay was carried out using human colon carcinoma HT29 cells. After 1 h of incubation, the amount of TOPO I linked to DNA was significantly increased by nevadensin (500 µM), why nevadensin was characterized as TOPO I poison. However, no effects on TOPO IIα were detected in the cellular test system. As a subsequent cellular response to TOPO I poisoning, a highly significant increase of DNA damage after 2 h and a decrease of cell viability after 48 h at the same concentration range were found. Furthermore, after 24 h of incubation a G2/M arrest was observed at concentrations ≥ 100 µM by flow cytometry. The analysis of cell death revealed that nevadensin induces the intrinsic apoptotic pathway via activation of caspase-9 and caspase-3. The results suggest that cell cycle disruption and apoptotic events play key roles in the cellular response to TOPO I poisoning caused by nevadensin in HT29 cells.

Butyrate Alters Pyruvate Flux and Induces Lipid Accumulation in Cultured Colonocytes

Butyrate is considered the primary energy source of colonocytes and has received wide attention due to its unique health benefits. Insight into the mechanistic effects of butyrate on cellular and metabolic function relies mainly on research in in-vitro-cultured cells. However, cells in culture differ from those in vivo in terms of metabolic phenotype and nutrient availability. For translation, it is therefore important to understand the impact of different nutrients on the effects of butyrate. We investigated the metabolic consequences of butyrate exposure under various culturing conditions, with a focus on the interaction between butyrate and glucose. To investigate whether the effects of butyrate were different between cell­­s with high and low mitochondrial capacity, we cultured HT29 cells under either low- (0.5 mM) or high- (25 mM) glucose conditions. Low-glucose culturing increased the mitochondrial capacity of HT29 cells compared to high-glucose (25 mM) cultured HT29 cells. Long-term exposure to butyrate did not alter mitochondrial bioenergetics, but it decreased glycolytic function, regardless of glucose availability. In addition, both high- and low-glucose-grown HT29 cells showed increased lipid droplet accumulation following long-term butyrate exposure. Acute exposure of cultured cells (HT29 and Caco-2) to butyrate increased their oxygen consumption rate (OCR). A simultaneous decrease in extracellular acidification rate (ECAR) was observed. Furthermore, in the absence of glucose, OCR did not increase in response to butyrate. These results lead us to believe that butyrate itself was not responsible for the observed increase in OCR, but, instead, butyrate stimulated pyruvate flux into mitochondria. Indeed, blocking of the mitochondrial pyruvate carrier prevented a butyrate-induced increase in oxygen consumption. Taken together, our results indicate that butyrate itself is not oxidized in cultured cells but instead alters pyruvate flux and induces lipid accumulation.

Nanoparticle-Encapsulated Camptothecin: Epigenetic Modulation in DNA Repair Mechanisms in Colon Cancer Cells

Molecular crosstalk between the cellular epigenome and genome converge as a synergistic driver of oncogenic transformations. Besides other pathways, epigenetic regulatory circuits exert their effect towards cancer progression through the induction of DNA repair deficiencies. We explored this mechanism using a camptothecin encapsulated in β-cyclodextrin–EDTA–Fe3O4 nanoparticles (CPT-CEF)-treated HT29 cells model. We previously demonstrated that CPT-CEF treatment of HT29 cells effectively induces apoptosis and cell cycle arrest, stalling cancer progression. A comparative transcriptome analysis of CPT-CEF-treated versus untreated HT29 cells indicated that genes controlling mismatch repair, base excision repair, and homologues recombination were downregulated in these cancer cells. Our study demonstrated that treatment with CPT-CEF alleviated this repression. We observed that CPT-CEF exerts its effect by possibly affecting the DNA repair mechanism through epigenetic modulation involving genes of HMGB1, APEX1, and POLE3. Hence, we propose that CPT-CEF could be a DNA repair modulator that harnesses the cell’s epigenomic plasticity to amend DNA repair deficiencies in cancer cells.

Exploring the cytotoxic mechanisms of Pediocin PA-1 towards HeLa and HT29 cells by comparison to known bacteriocins: Microcin E492, enterocin heterodimer and Divercin V41

The purpose of this study was to explore potential mechanisms of cytotoxicity towards HeLa and HT29 cells displayed by Pediocin PA-1. We did this by carrying out sequence alignments and 3D modelling of related bacteriocins which have been studied in greater detail: Microcin E492, Enterocin AB heterodimer and Divercin V41. Microcin E492 interacts with Toll-Like Receptor 4 in order to activate an apoptosis reaction, sequence alignment showed a high homology between Pediocin PA-1 and Microcin E492 whereas 3D modelling showed Pediocin PA-1 interacting with TLR-4 in a way reminiscent of Microcin E492. Furthermore, Pediocin PA-1 had the highest homology with the Enterocin heterodimer, particularly chain A; Enterocin has also shown to cause an apoptotic response in cancer cells. Based on this we are led to strongly believe Pediocin PA-1 interacts with TLRs in order to cause cell death. If this is the case, it would explain the difference in cytotoxicity towards HeLa over HT29 cells, due to difference in expression of particular TLRs. Overall, we believe Pediocin PA-1 exhibits a dual effect which is dose dependant, like that of Microcin. Unfortunately, due to the COVID-19 pandemic, we were unable to carry out experiments in the lab, and the unavailability of important data meant we were unable to provide and validate out solid conclusions, but rather suggestions. However, bioinformatic analysis is still able to provide information regarding structure and sequence analysis to draw plausible and evidence based conclusions. We have been able to highlight interesting findings and how these could be translated into future research and therapeutics in order to improve the quality of treatment and life of cancer patients.

NanI Sialidase Contributes to the Growth and Adherence of Clostridium perfringens Type F Strain F4969 in the Presence of Adherent Mucus

Clostridium perfringens type F strains causing non-foodborne human gastrointestinal diseases (NFD) typically produce NanI sialidase as their major secreted sialidase. Type F NFDs can persist for several weeks, indicating their pathogenesis involves intestinal colonization, including vegetative cell growth and adherence, with subsequent sporulation that fosters enterotoxin production and release. We previously reported that NanI contributes to type F NFD strain adherence to, and growth using, Caco-2 cells. However, Caco-2 cells make minimal amounts of mucus, which is significant because the intestines are coated with adherent mucus. Therefore, it was important to assess if NanI contributes to the growth and adherence of type F NFD strains in the presence of adherent mucus. Consequently, the current study first demonstrated greater growth of nanI -carrying vs. non- nanI -carrying type F strains in the presence of HT29-MTX-E12 cells, which produce an adherent mucus layer, vs. their parental HT29 cells, which make minimal mucus. Demonstrating the specific importance of NanI for this effect, type F NFD strain F4969 or a complementing strain grew and adhered better than an isogenic nanI null mutant in the presence of HT29-MTX-E12 cells vs HT29 cells. Those effects involved mucus production by MTX-E12 cells since mucus reduction using N-acetyl-cysteine reduced F4969 growth and adherence. Consistent with those in vitro results, NanI contributed to growth of F4969 in the mouse small intestine. By demonstrating a growth and adherence role for NanI in the presence of adherent mucus, these results further support NanI as a potential virulence factor during type F NFDs.