Increased ENT2 Expression and Its Association With Altered Purine Metabolism in Different Stages of Colorectal Cancer Cell Lines

Background Colorectal cancer (CRC) is one of the most prevalent malignant cancers worldwide. Although the purine metabolism pathway is known to be vital for cancer cells survival mechanism, not much is known on ENT2 role in CRC development and its association with purine metabolites. Hence this study is aimed to determine the level of hypoxanthine phosphoribosyl transferase (HPRT), hypoxanthine, uric acid (UA), and the activity of xanthine oxidase (XO) and relate the findings with the ENT2 expression level in different CRC stages. Methods and results Normal colon cell line; CCD-841CoN and a panel of human CRC cell lines; SW480, HCT15 and HCT116, representing different CRC stages; Dukes’ B, C, and D respectively, have been used to measure HPRT, hypoxanthine/xanthine, UA levels and the activity of XO by biochemical assays. The level of ENT2 gene expression was also performed by qRT-PCR. The levels of HPRT, hypoxanthine were significantly higher (P< 0.05), while XO and UA were lower (P< 0.05) in all CRC stages as compared to the normal colon cells. Furthermore, ENT2 expression was found to be increased in all CRC stages. Despite having the highest level of HPRT and hypoxanthine, ENT2 level is lower in Dukes' D when compared to Dukes' B and C. Conclusion The rate of salvage pathway is increased in CRC development as indicated by the elevated levels of HPRT and hypoxanthine in different CRC stages. Increase ENT2 expression implies its importance in assisting hypoxanthine uptake. This step is vital in order to increase DNA synthesis via hypoxanthine recycling. Thus, ENT2 may be a potential marker in therapeutic development.

Potential Antioxidant and Anticancer Activities of Secondary Metabolites of Nostoc linckia Cultivated under Zn and Cu Stress Conditions

The objective of the present study is to determine the antioxidant and anticancer activities of Nostoc linckia extracts cultivated under heavy metal stress conditions (0.44, 0.88, and 1.76 mg/L for zinc and 0.158, 0.316, 0.632 mg/L for copper). Phycobiliprotein, phenolic compounds, flavonoids, and tannins were measured. Active ingredients of extracts were evaluated by GC-mass spectroscopy. The obtained results revealed that higher zinc and copper concentrations showed growth inhibition while 0.22 mg/L (Zn) and 0.079 mg/L (Cu) enhanced growth, reaching its maximum on the 25th day. Increases in catalase, lipids peroxidation, and antioxidants, as well as tannins and flavonoids, have been induced by integration of 0.88 mg/L (Zn) and 0.316 mg/L (Cu). Elevation of Zn concentration induced augmentation of antioxidant activity of crude extract (DPPH or ABTS), with superior activity at 0.44 mg/L zinc concentration (81.22%). The anticancer activity of Nostoc linckia extract (0.44 mg/L Zn) tested against four cancer cell lines: A549, Hela, HCT 116, and MCF-7. The extract at 500 µg/mL appeared the lowest cell viability of tested cell lines. The promising extract (0.44 mg/L Zn) recorded the lowest cell viability of 25.57% in cervical cell line, 29.74% in breast cell line, 33.10% in lung cell line and 34.53% in the colon cell line. The antioxidant active extract showed significant stability against pH with attributed increase in antioxidant activity in the range between 8–12. The extract can be used effectively as a natural antioxidant and anticancer after progressive testing.

Assessing the Biocompatibility of Multi-Anchored Glycoconjugate Functionalized Iron Oxide Nanoparticles in a Normal Human Colon Cell Line CCD-18Co

We have previously demonstrated that iron oxide nanoparticles with dopamine-anchored heterobifunctional polyethylene oxide (PEO) polymer, namely PEO-IONPs, and bio-functionalized with sialic-acid specific glycoconjugate moiety (Neu5Ac(α2-3)Gal(β1-4)-Glcβ-sp), namely GM3-IONPs, can be effectively used as antibacterial agents against target Escherichia coli. In this study, we evaluated the biocompatibility of PEO-IONPs and GM3-IONPs in a normal human colon cell line CCD-18Co via measuring cell proliferation, membrane integrity, and intracellular adenosine triphosphate (ATP), glutathione GSH, dihydrorhodamine (DHR) 123, and caspase 3/7 levels. PEO-IONPs caused a significant decrease in cell viability at concentrations above 100 μg/mL whereas GM3-IONPs did not cause a significant decrease in cell viability even at the highest dose of 500 μg/mL. The ATP synthase activity of CCD-18Co was significantly diminished in the presence of PEO-IONPs but not GM3-IONPs. PEO-IONPs also compromised the membrane integrity of CCD-18Co. In contrast, cells exposed to GM3-IONPs showed significantly different cell morphology, but with no apparent membrane damage. The interaction of PEO-IONPs or GM3-IONPs with CCD-18Co resulted in a substantial decrease in the intracellular GSH levels in a time- and concentration-dependent manner. Conversely, levels of DHR-123 increased with IONP concentrations. Levels of caspase 3/7 proteins were found to be significantly elevated in cells exposed to PEO-IONPs. Based on the results, we assume GM3-IONPs to be biocompatible with CCD-18Co and could be further evaluated for selective killing of pathogens in vivo.

Targeting the hallmarks of cancer: the effects of silibinin on proliferation, cell death, angiogenesis, and migration in colorectal cancer

Background Silibinin, as a chemopreventive agent, has shown anti-cancer efficacy against different types of cancers. In the present study, we investigated the anti-cancer activities of silibinin on CT26 mouse colon cell line. Methods CT26 cells were treated with different concentrations of silibinin. To examine the cytotoxic effect of silibinin on proliferation, apoptosis, autophagy, angiogenesis, and migration, MTT, colony-forming assay, Annexin V/PI flow cytometry, RT-qPCR, and Scratch assay were used. Results Silibinin was found to significantly reduce CT26 cells survival. Furthermore, silibinin strongly induced apoptosis and autophagy by up-regulating the expression of Bax, Caspase-3, Atg5, Atg7 and BECN1 and down-regulating Bcl-2. Silibinin considerably down-regulated the expression of COX-2, HIF-1α, VEGF, Ang-2, and Ang-4 as well as the expression of MMP-2, MMP-9, CCR-2 and CXCR-4. Conclusions The present study revealed that silibinin shows anticancer activities by targeting proliferation, cell survival, angiogenesis, and migration of CT26 cells.

Oxidative stress induced by tBHP in human normal colon cells by label free Raman spectroscopy and imaging. The protective role of natural antioxidants in the form of β-carotene

The present study aimed to investigate the protective effect of β-carotene on the oxidative stress injury of human normal colon cell line CCD-18Co triggered by tert-butyl hydroperoxide (tBHP).

Differential gene expression and functional pathway enrichment in colon cell line CCD 841 CoN (CRL-1790) transfected with miR-mimics miR-18b, miR-142-3p, miR-155, and miR-890

This report is a bioinformatic analysis of samples from NCBI GEO database series GS132501. It includes previously un-reported data for miR-mimic over-expression of the miRNAs miR-18b, miR-155, miR-142-3p, and miR-890 in the ATCC CRL-1790 cell line. Data analysis was performed using Nanostring nSolver 4.0 and Advanced Analysis Module 2.0 plugin (Nanostring MAN-10030-03). Bioinformatic methods utilized include pathway scoring, differential expression (DE), and gene-set enrichment (GSE) analyses. Findings, with full supplementary data, provide a community resource for effects of dysregulation of these miRNAs in a colon ‘epithelial-like’ cell line.

Arabinoxylan-Based Particles: In Vitro Antioxidant Capacity and Cytotoxicity on a Human Colon Cell Line

Background and objectives: Arabinoxylans (AX) can gel and exhibit antioxidant capacity. Previous studies have demonstrated the potential application of AX microspheres as colon-targeted drug carriers. However, the cytotoxicity of AX gels has not been investigated so far. Therefore, the aim of the present study was to prepare AX-based particles (AXM) by coaxial electrospraying method and to investigate their antioxidant potential and cytotoxicity on human colon cells. Materials and Methods: The gelation of AX was studied by monitoring the storage (G′) and loss (G′′) moduli. The morphology of AXM was evaluated using optical and scanning electron microscopy (SEM). The in vitro antioxidant activity of AX before and after gelation was measured using the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) methods. In addition, the effect of AX and AXM on the proliferation of human colon cells (CCD 841 CoN) was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: The final G′ and G′′ values for AX gels were 293 and 0.31 Pa, respectively. AXM presented spherical shape and rough surface with a three-dimensional and porous network. The swelling ratio and mesh size of AXM were 35 g water/g AX and 27 nm, respectively. Gelation decreased the antioxidant activity of AX by 61–64 %. AX and AXM did not affect proliferation or show any toxic effect on the normal human colon cell line CCD 841 CoN. Conclusion: The results indicate that AXM could be promising biocompatible materials with antioxidant activity.

Deciphering the Mechanism of Action Involved in Enhanced Suicide Gene Colon Cancer Cell Killer Effect Mediated by Gef and Apoptin

Despite the great advances in cancer treatment, colorectal cancer has emerged as the second highest cause of death from cancer worldwide. For this type of tumor, the use of suicide gene therapy could represent a novel therapy. We recently demonstrated that co-expression of gef and apoptin dramatically inhibits proliferation of the DLD-1 colon cell line. In the present manuscript, we try to establish the mechanism underlying the enhanced induction of apoptosis by triggering both gef and apoptin expression in colon tumor cells. Scanning microscopy reveals that simultaneous expression of gef and apoptin induces the apparition of many “pores” in the cytoplasmic membrane not detected in control cell lines. The formation of pores induced by the gef gene and accentuated by apoptin results in cell death by necrosis. Moreover, we observed the presence of apoptotic cells. Performing protein expression analysis using western blot, we revealed an activation of mitochondrial apoptosis (increased expression of Pp53, cytochrome c, Bax, and caspase 9) and also the involvement of the extrinsic pathway through caspase 8activation. In conclusion, in this manuscript we demonstrate for the first time that the extrinsic pathway of apoptosis and pore formation is also involved in the cell death caused by the co-expression of the gef and apoptin genes. Our results suggest that co-expression of gef and apoptin genes induces an increase in post-apoptotic necrotic cell death and could be a valuable tool in the design of new antitumor strategies focused on the enhancement of the immune response against cancer cell death.

Extraction, Identification and Biological Activities of Saponins in Sea Cucumber Pearsonothuria graeffei

Aims and Objectives: Secondary metabolism in marine organisms produces a diversity of biologically important natural compounds that are not present in terrestrial species. Sea cucumbers belong to the invertebrate Echinodermata and are famous for their nutraceutical, medical and food values. They are known for possession triterpenoid glycosides (saponins) with various ecological roles. The current work aimed to separate, identify and test various biological activities (antibacterial, antifungal, antileishmanial and anticancer properties) of saponins produced by the holothurian Pearsonothuria graeffei from the Red Sea, Egypt. Materials and Methods: The structures were identified by 1D and 2D NMR (1H, 13C, TOCSY, COSY, HSQC, HMBC, and ROESY) experiments and acid hydrolysis. The crude and purified fractions was analyzed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)/MS to identify saponins and characterize their molecular structures. Partially purified fraction, mainly containing compounds 1 and 2, was screened for its antifungal activity against three clinical isolates of Candida albicans (Candida 580 (1), Candida 581(2) and Candida MEO47228. Antileishmanial activity against Leishmania major and toxicity on colon cell-line were also evaluated. Results: Two lanostane type sulfated triterpene monoglycosides were isolated from the Holothurian Pearsonothuria graeffei from the Red Sea, Egypt. Holothurin A (1) and echinoside A (2) triterpene saponins were separated by reversed phase semi-preparative HPLC. LC50 values (µg/mL); calculated for the fraction containing saponins 1 and 2 as major constituents; against Candida albicans, Leishmania major and colon cell-line were 10, 20 and 0.50, respectively. Conclusion: Consequently, this study demonstrated the potential use of sea cucumber Pearsonothuria graeffei not only as appreciated functional food or nutraceuticals but also as the source of functional ingredients for pharmaceutical products with antifungal, antileishmanial and anticancer properties.