Colorectal Carcinoma HCT116 Cells Research Articles

267 - Biological Characterization of the Anti-Tumor Properties of Novel Dual-Target Inhibitors of Dihydrofolate Reductase and Thioredoxin Reductase

Drug resistance to chemotherapy is one reason why many cancer treatments have failed. To combat this problem, a strategy of simultaneously targeting several vital systems of the cancer cell could be effective. Dihydrofolate reductase (DHFR) and thioredoxin reductase (TrxR) are enzymes belonging to two unrelated cellular pathways that are vital for cancer cell growth. Novel dual target inhibitors of DHFR and TrxR had been synthesized and lead compounds displaying strong anti-proliferative activities against HCT116 colorectal and MCF-7 breast carcinoma cells that were correlated to their in vitro DHFR and TrxR inhibitory potencies were identified. Further biological characterization work demonstrated that in HCT116 cells, the lead compounds exhibited inhibitory effects against DHFR and TrxR activities and brought about induction of apoptosis in a dose-dependent manner. Cell viability assays carried out on wild-type and methotrexate-resistant HCT116 cells had revealed that the lead DHFR-TrxR dual targeting compounds were effective in inhibiting growth in the resistant cell line, suggesting that the novel compounds could potentially serve as clinical candidates in anticancer therapies.

Photo-bioreduction of Ag+ ions towards the generation of multifunctional silver nanoparticles: Mechanistic perspective and therapeutic potential

In this article, light induced plant extract mediated one pot synthesis of silver nanoparticles (AgNPs) has been demonstrated and potential mechanistic insight in the synthesis has been investigated. Bioactive molecules containing medicinal plant Cassytha filiformis has been explored for the synthesis of silver nanoparticles. The as-synthesized silver nanoparticles were characterized by various analytical techniques including Ultraviolet–visible spectroscopy (UV–Vis), High Resolution Transmission Electron Microscopy (HR-TEM), Dynamic Light Scattering (DLS) and Fourier Transform Infrared Spectroscopy (FT-IR). Among different light sources (sunlight, room light, UV) applied the sunlight was found to be efficient external stimuli to induce rapid synthesis of AgNPs at room temperature. Modified DPPH assay indicated that polyphenolic compounds were most likely involved in the synthesis of AgNPs. Possible molecule responsible for the synthesis of AgNPs was identified, purified and characterized. Potential biomedical applications such as antibacterial, antifungal and anticancer activities of AgNPs have been evaluated. Irrespective of nature of pathogenic strains nanoparticles exhibited significant antibacterial activities against Gram positive (Streptococcus aureus) and Gram negative (Escherichia coli) bacterial pathogens. It showed higher activity on E. coli than on S. aureus. Distinct antifungal activity (MIC=5.244μg/ml) and remarkable anticancer activity (IC50=10μg/ml) was found against Candida albicans and HCT116 (colorectal carcinoma) cells, respectively. Taken together, these findings suggested that light induced plant generated silver nanoparticles could be used for various biomedical purposes.

SiRNA-Mediated Silencing of HMGA2 Induces Apoptosis and Cell Cycle Arrest in Human Colorectal Carcinoma.

Overexpression of HMGA2, known as small non-histone chromosomal protein, is associated with progression of various tumors, including colorectal cancers. The aim of this study was to investigate the effect of a specific HMGA2 siRNA on apoptosis and cell cycle of HCT-116 (colorectal carcinoma) cells. The cells were transfected with siRNAs using a transfection reagent. The cytotoxic effects of HMGA2 siRNA on colorectal carcinoma cells were determined using MTT assay. Relative HMGA2 mRNA and protein levels were measured by QRT-PCR and western blotting, respectively. Apoptosis was measured by a TUNEL test based on labeling of DNA strand breaks. We also evaluated caspase-3, caspase-8, and caspase-9 expression by QRTPCR to determine which pathway is involved in apoptosis. Cell cycle was assessed by FACS and cell cycle analysis using PI DNA staining. HMGA2 siRNA significantly reduced both mRNA and protein expression levels 48 h after transfection and dose-dependent manner in colorectal carcinoma cells. We also showed that the silencing of HMGA2 led to the induction of apoptosis through intrinsic pathway and cell cycle arrest in G2/M phases of interphase in HCT-116 cells in vitro. These results propose that HMGA2 might play an important role in the progression of colorectal carcinoma and might be a potential therapeutic target for trigger apoptosis and cell cycle arrest in colorectal carcinoma.

Long non-coding RNA AFAP1-AS1 facilitates tumor growth and promotes metastasis in colorectal cancer.

Background and objectiveLong non-coding RNAs can regulate tumorigenesis of various cancers. Dys-regulation of lncRNA-AFAP1-AS1 has not been studied in colorectal carcinoma (CRC). This study was to examine the function involvement of AFAP1-AS1 in tumor growth and metastasis of CRC.MethodsRelative expression of AFAP1-AS1 in CRC tissues and CRC cells lines was determined using quantitative real-time PCR (qRT-PCR). Functional involvement of AFAP1-AS1 in tumor proliferation and metastasis was evaluated in AFAP1-AS1-specific siRNA-treated CRC cells and in CRC cell xenograft. Expression of epithelial-mesenchymal transition (EMT)-related gene expression was determined using western blot.ResultsRelative expression of AFAP1-AS1 was significantly elevated in CRC tissues and CRC HCT116 and SW480 cell lines. AFAP1-AS1 knock-down suppressed SW480 cell proliferation, colony formation, migration and invasion. Also AFAP1-AS1 knock-down inhibited tumor metastasis-associated genes expression in terms of EMT. This carcinostatic action by AFAP1-AS1 knock-down was further confirmed by suppression of tumor formation and hepatic metastasis of CRC cells in nude mice.ConclusionlncRNA-AFAP1-AS1 knock-down exhibits antitumor effect on colorectal carcinoma in respects of suppression of cell proliferation and metastasis of cancer cells.

Abstract 80: Programmatic re-splicing of the hypoxia transcriptome regulates the DNA damage response

Abstract Background Hypoxia occurs within the majority of solid tumors. It has multiple impacts on tumor biology including angiogenesis, suppression of immune reactivity, enhanced receptor tyrosine kinase signaling, down regulation of DNA repair pathways, promotion of pro-survival phenotypes and increased proclivity for invasion and metastasis. Levels of hypoxia vary between and within tumors, correlate with patient outcomes, and can lead to differences in response to therapy. Despite considerable advances, our understanding of the regulatory processes that control these diverse and heterogeneous changes in cancer phenotype remain incomplete. A better understanding of how they are mediated will inform strategies for personalized medicine and promise to substantially advance our understanding of tumor heterogeneity. Methods Until recently, techniques for RNA-expression profiling have lacked the resolution necessary to detect genome wide changes in splicing. We exploited the increased precision offered by deep sequencing to investigate genome-wide remodelling of transcript architectures in both cell line and tumor samples. Using novel computational strategies we generated sample-specific gene models from RNA sequencing data generated over a timecourse of HCT116 colorectal carcinoma cells following a switch to 1% oxygenation and used these data to identify widespread changes in splicing. We applied similar strategies to raw RNA-sequencing data derived from TCGA. Results We detected the expression of thousands of novel isoforms and a systematic, pathway-dependent switch to the expression of noncoding transcripts at multiple genes, including many constituents of the Fanconi Anemia, nucleotide excision- and double strand break repair pathways. Of particular note were HDAC6 and 53BP1: critical regulators of DNA damage response. Both loci exhibited a hypoxia-dependent expression of retained-intron transcripts and a concomitant decline in protein levels, thus providing a novel and unanticipated mechanism by which hypoxia can drive genetic instability. Predictions made using our cell line model were recapitulated in a large cohort of 458 colorectal carcinomas derived from TCGA. Validation of the cell line data revealed, for the first time, a strong signature of isoform switching in which a widespread transition to noncoding expression is associated with poor patient outcome. These data were derived from independent patient biopsies sequenced by others, thus demonstrating the robustness of our findings. Conclusions These data therefore have clear, substantial, and immediate implications both for our understanding of the basic aetiology of solid tumors and for the development of RNA-based signatures and biomarkers in diagnostics and personalized medicine. Citation Format: Crispin Miller, Danish Memon, Keren Dawson, Christopher Smowton, Wei Xing, Caroline Dive. Programmatic re-splicing of the hypoxia transcriptome regulates the DNA damage response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 80.

Pogostone induces autophagy and apoptosis involving PI3K/Akt/mTOR axis in human colorectal carcinoma HCT116 cells

Ethnophamacological relevancePogostemon cablin is a medicinal herb widely used to treat gastrointestinal diseases in many Asian countries. Pogostone is an important constituent of Pogostemon cablin, and possesses various bioactivitys. In this study, we performed to investigate the anti-colorectal tumor property of Pogostone by inducing aurophagy and apoptosis in human colorectal cancer cells, and to define the potential molecular mechanisms. Materials and methodsIn vitro, The anti-tumor activity of pogostone was assessed using MTT assay. Autophagy was monitored by transmission electron microscopy observation and mRFP-GFP-LC3 fluorescence analysis in colorectal tumor cell line. Apoptosis was measured by flow cytometry and annexinV-FITC/PI staining. The protein expressions or activition of LC3-Ⅱ, AKT, mTOR, caspase-3 and caspase-7 were detected through western blotting. In vivo, the anti-tumor effect of pogostone was tested with HCT116 colorectal tumor cells transplantation tumor model. The expression of Ki-67 was determined by Immunohistochemistry staining and the apoptosis was evaluated using TUNEL assay. ResultsIn vitro, pogostone exhibits significant anti-tumor activity against human cancer cell lines, especially for HCT116 (18.7±1.93μg/ml). Transmission electron microscopy observation, mRFP-GFP-LC3 fluorescence analysis, flow cytometry and assay and western blotting detection revealed that the anti-colorectal tumor activity of pogostone was dependent on inducing autophagy and apoptosis through up-regulating the expression of LC3-Ⅱ, cleaved caspase-7 and caspase-3, and decreasing the phosphorylation of AKT/mTOR. In vivo, 150mg/kg pogostone inhibited the HCT116 tumor growth in immunodeficient mice with an inhibitory rate of 43.3%, decreased the expression of Ki67, and induced apoptosis in three days. ConclusionPogostone showed anti-colorectal tumor effects by inducing autophagy and apoptosis involving PI3K/Akt/mTOR axis. Thus, pogostone may be a promising lead compound to be further developed for cancer therapy.

The UVB1 Vitamin D analogue inhibits colorectal carcinoma progression

Vitamin D has been shown to display a wide variety of antitumour effects, but their therapeutic use is limited by its severe side effects. We have designed and synthesized a Gemini vitamin D analogue of calcitriol (UVB1) which has shown to display antineoplastic effects on different cancer cell lines without causing hypercalcemia. The aim of this work has been to investigate, by employing in silico, in vitro, and in vivo assays, whether UVB1 inhibits human colorectal carcinoma progression. We demonstrated that UVB1 induces apoptotic cell death and retards cellular migration and invasion of HCT116 colorectal carcinoma cells. Moreover, the analogue reduced the tumour volume in vivo, and modulated the expression of Bax, E-cadherin and nuclear β-catenin in tumour animal tissues without producing toxic effects. In silico analysis showed that UVB1 exhibits greater affinity for the ligand binding domain of vitamin D receptor than calcitriol, and that several characteristics in the three-dimensional conformation of VDR may influence the biological effects. These results demonstrate that the Gemini vitamin D analogue affects the growth of the colorectal cancer and suggest that UVB1 is a potential chemotherapeutic agent for treatment of this disease.

The synthesis and biological evaluation of unsymmetrical 2,2-di(1H-indol-3-yl)-N-phenylacetamide derivatives

A mild and highly efficient method has been developed for the synthesis of unsymmetrical 2,2-di(1H-indol-3-yl)-N-phenylacetamide derivatives by the regioselective Friedel–Crafts alkylation of 2-hydroxy-2-(1H-indol-3-yl)-N-phenylacetamide derivatives with various indoles catalyzed by AlCl3 at room temperature in a short reaction time in high yields (up to 96%). All these compounds were screened for their antiproliferative activity against human colorectal carcinoma HCT116 cell line. The preliminary biological study showed that some of them exhibited moderate to good antiproliferative activity in vitro. Especially, compound 3e exerted the most powerful antiproliferative activity even better than the positive control MDM-2/p53 antagonist Nutlin-3a.

TRAIL-Induced Caspase Activation Is a Prerequisite for Activation of the Endoplasmic Reticulum Stress-Induced Signal Transduction Pathways.

It is well known that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis can be initially triggered by surface death receptors (the extrinsic pathway) and subsequently amplified through mitochondrial dysfunction (the intrinsic pathway). However, little is known about signaling pathways activated by the TRAIL-induced endoplasmic reticulum (ER) stress response. In this study, we report that TRAIL-induced apoptosis is associated with the endoplasmic reticulum (ER) stress response. Human colorectal carcinoma HCT116 cells were treated with TRAIL and the ER stress-induced signal transduction pathway was investigated. During TRAIL treatment, expression of ER stress marker genes, in particular the BiP (binding immunoglobulin protein) gene, was increased and activation of the PERK (PKR-like ER kinase)-eIF2α (eukaryotic initiation factor 2α)-ATF4 (activating transcription factor 4)-CHOP (CCAAT-enhancer-binding protein homologous protein) apoptotic signal transduction pathway occurred. Experimental data from use of a siRNA (small interfering RNA) technique, caspase inhibitor, and caspase-3-deficient cell line revealed that TRAIL-induced caspase activation is a prerequisite for the TRAIL-induced ER stress response. TRAIL-induced ER stress was triggered by caspase-8-mediated cleavage of BAP31 (B cell receptor-associated protein 31). The involvement of the proapoptotic PERK-CHOP pathway in TRAIL-induced apoptosis was verified by using a PERK knockout (PERK(-/-)) mouse embryo fibroblast (MEF) cell line and a CHOP(-/-) MEF cell line. These results suggest that TRAIL-induced the activation of ER stress response plays a role in TRAIL-induced apoptotic death.

Facile Synthesis of Large‐Pore Bicontinuous Cubic Mesoporous Silica Nanoparticles for Intracellular Gene Delivery

AbstractMesoporous silica nanoparticles (MSNs) have promising biomedical applications; however the synthesis of MSNs with a bicontinuous cubic Ia3d mesostructure and large pore size has been a long‐standing challenge. Herein we report a systematic study on the synthesis of KIT‐6 type MSNs (KIT‐6‐MSNs) using fluorocarbon‐hydrocarbon mixed surfactants as templates and ethanol as a co‐solvent. KIT‐6‐MSNs have a pore size of 9 nm and a particle size in the range of 200–400 nm. The role of ethanol in the synthesis of KIT‐6‐MSNs is crucial because ethanol increases the miscibility of fluorocarbon and hydrocarbon surfactants, thereby increasing the packing parameter and favoring the formation of an Ia3d structure. Excessive fluorocarbon surfactants help the formation of nanosized morphology. After hydrophobic modification, the large‐pore KIT‐6‐MSNs show a high loading capacity towards genetic molecules and successfully deliver the survivin siRNA into a human colorectal carcinoma HCT116 cell line, knocking down the gene expression associated with cancer cell survival and proliferation.

Evidence that 2-hydroxyglutarate is not readily metabolized in colorectal carcinoma cells.

BackgroundTwo-hydroxyglutarate (2HG) is present at low concentrations in healthy mammalian cells as both an L and D enantiomer. Both the L and D enantiomers have been implicated in regulating cellular physiology by mechanisms that are only partially characterized. In multiple human cancers, the D enantiomer accumulates due to gain-of-function mutations in the enzyme isocitrate dehydrogenase (IDH) and has been hypothesized to drive malignancy through mechanisms that remain incompletely understood. While much attention has been dedicated to identifying the route of 2HG synthesis, the metabolic fate of 2HG has not been studied in detail. Yet the metabolism of 2HG may have important mechanistic consequences influencing cell function and cancer pathogenesis, such as modulating redox potential or producing unknown products with unique modes of action.ResultsBy applying our isotope-based metabolomic platform, we unbiasedly and comprehensively screened for products of L- and D-2HG in HCT116 colorectal carcinoma cells harboring a mutation in IDH1. After incubating HCT116 cells in uniformly 13C-labeled 2HG for 24 h, we used liquid chromatography/mass spectrometry to track the labeled carbons in small molecules. Strikingly, we did not identify any products of 2HG metabolism from the thousands of metabolomic features that we screened. Consistent with these results, we did not detect any significant changes in the labeling patterns of tricarboxylic acid cycle metabolites from wild type or IDH1 mutant cells cultured in 13C-labeled glucose upon the addition of L, D, or racemic mixtures of 2HG. A more sensitive, targeted analysis revealed trace levels of isotopic enrichment (<1 %) in some central carbon metabolites from 13C-labeled 2HG. However, we found that cells do not deplete 2HG from the media at levels above our detection limit over a 48 h time period.ConclusionsTaken together, we conclude that 2HG carbon is not readily transformed in the HCT116 cell line. These data indicate that the phenotypic alterations induced by 2HG are not a result of its metabolic products.

The role of interleukin-8 (CXCL8) and CXCR2 in acquired chemoresistance of human colorectal carcinoma cells HCT116.

Colorectal cancer is one of the most common malignant diseases and is a leading cause of cancer mortality in the Western world. Primary or acquired resistance to chemotherapeutic drugs is a common phenomenon which causes a failure in cancer treatment. A diverse range of molecular mechanisms has been implicated in drug resistance: DNA damage repair, alterations in drug metabolism, mutation of drug targets, increased rates of drug efflux, and activation of survival signaling pathways. The aim of this study was to investigate the expression of CXCL8-CXCR1/2 pathway, its impact on cell proliferation and cytokine expression in human colorectal carcinoma HCT116 cells, and their chemotherapy-resistant subline. We found that IL-1 alpha stimulates the production of CXCL8 through IL-1 receptor signaling. Our data indicate that CXCL8 is upregulated in chemoresistant subline of colorectal cancer cells HCT116, and modulation of CXCR2 pathway can be a target for proliferation inhibition of chemoresistant colorectal cancer cells.

Purification, partial characterization and antitumor effect of an exopolysaccharide from Rhizopus nigricans

In this study, a homogeneous exopolysaccharide (EPS1-1) was purified from the fermentation broth of Rhizopus nigricans. EPS1-1 was composed of glucose, mannose, galactose and fructose in the molar ratio of 5.89:3.64:3.20:1.00 with weight average molecular weight of 9.7×103g/mol. EPS1-1 could significantly inhibit proliferation of human colorectal carcinoma HCT-116 cells in vitro. EPS1-1 also induced S phase cell cycle arrest and increased sub-G0/G1 population, a hallmark of apoptosis. The results of morphological characterization and flow cytometry showed that EPS1-1 induced apoptotic cell death in HCT-116 cells. EPS1-1 caused dissipation of mitochondrial membrane potential, accumulation of reactive oxygen species, up-regulation of Bax and p53 mRNA expression and down-regulation of Bcl-2 mRNA expression, which suggested that mitochondrial pathway was involved in the EPS1-1-induced apoptosis. These findings bring new insights into the potential use of EPS1-1 as antitumor drug against human colorectal carcinoma.

Construction of a plasmid coding for green fluorescent protein tagged cathepsin L and data on expression in colorectal carcinoma cells.

The endo-lysosomal cysteine cathepsin L has recently been shown to have moonlighting activities in that its unexpected nuclear localization in colorectal carcinoma cells is involved in cell cycle progression (Tamhane et al., 2015) [1]. Here, we show data on the construction and sequence of a plasmid coding for human cathepsin L tagged with an enhanced green fluorescent protein (phCL-EGFP) in which the fluorescent protein is covalently attached to the C-terminus of the protease. The plasmid was used for transfection of HCT116 colorectal carcinoma cells, while data from non-transfected and pEGFP-N1-transfected cells is also shown. Immunoblotting data of lysates from non-transfected controls and HCT116 cells transfected with pEGFP-N1 and phCL-EGFP, showed stable expression of cathepsin L-enhanced green fluorescent protein chimeras, while endogenous cathepsin L protein amounts exceed those of hCL-EGFP chimeras. An effect of phCL-EGFP expression on proliferation and metabolic states of HCT116 cells at 24 h post-transfection was observed.

The 1,3-diaryltriazenido(p-cymene)ruthenium(II) complexes with a high in vitro anticancer activity

1,3-Diaryltriazenes (1) were let to react with [RuCl2(p-cymene)]2 in the presence of trimethylamine to give neutral 1,3-diaryltriazenido(p-cymene)ruthenium(II) complexes, [RuCl(p-cymene)(ArNNNAr)] (2). The molecular composition of the products 2 was confirmed by NMR spectroscopy and mass spectrometry. The structures of the selected complexes were confirmed by a single crystal X-ray analysis. All triazenido-ruthenium complexes were highly cytotoxic against human cervical carcinoma HeLa cells with IC50 below 6μM, as determined by a spectrophotometric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) method. The most active was [RuCl(p-cymene)(ArNNNAr)] (Ar=4-Cl-3-(CF3)-C6H3) (2g) with IC50 of 0.103±0.006μM. In comparison with the data for the non-coordinated triazenes 1, the triazenido-ruthenium complexes 2 exhibited up to 560-times higher activity. Three selected complexes were highly cytotoxic also against several tumor cell lines: laryngeal carcinoma HEp-2 cells and their drug-resistant HEp-2 subline (7T), colorectal carcinoma HCT-116 cells, lung adenocarcinoma H460 cells, and mammary carcinoma MDA-MB-435 cells. The compounds 2g and [RuCl(p-cymene)(ArNNNAr)] (Ar=4-I-C6H4) (2j) were similarly cytotoxic against parental and drug-resistant cells. Time and dose dependent accumulation of the cells in the S phase of the cell cycle was induced by the compound 2g, triggering apoptosis. Our preliminary results indicate triazenido-ruthenium complexes as promising anticancer drug candidates.

Abstract 4480: Evaluation of anti-tumor enone-based bioactive compounds as specific thioredoxin reductase inhibitors

Abstract The thioredoxin system is a promising anti-cancer therapeutic target. It is one of the main cellular thiol antioxidant systems and comprises of thioredoxin, thioredoxin reductase and NADPH. In particular, thioredoxin reductase is an attractive target with susceptibility to inhibition by electrophilic compounds due to its nucleophilic selenocysteine residue in the C-terminal active center. In our laboratory, a series of enone-based compounds were synthesized and evaluated for their possible selectivity to inhibit thioredoxin reductase. To this end, the compounds were first screened for their in vitro thioredoxin reductase and glutathione reductase inhibitory activities, and their anti-proliferative activities were assessed by the MTT cell viability assay. The effects of selected analogs on the activities of thioredoxin reductase, thioredoxin and glutathione reductase were next studied. Finally, the molecular events linking thioredoxin reductase inhibition to apoptosis induction were elucidated. A number of the enone-based structural analogs were identified to posses anti-proliferative activities against HCT116 colorectal and MCF7 breast carcinoma cells at GI50 values in the low micromolar range, as well as inhibit activity of rat thioredoxin reductase with half-maximal inhibitory concentrations in the nanomolar range. Furthermore, these compounds displayed relatively weaker in vitro glutathione reductase inhibitory activities with half-maximal inhibitory concentrations in the micromolar range. In corroboration with results obtained from the in vitro enzyme activity assays, the identified lead analogs exhibited significant dose-dependent cellular thioredoxin reductase inhibition accompanied by a near negligible lack of effect on cellular thioredoxin and glutathione reductase activity. Conversely, the parent compound was found to not only inhibit cellular thioredoxin reductase activity, but also caused an inhibition of cellular thioredoxin and glutathione reductase activities. Finally, at a molecular level, the identified lead analogs that exhibited selective inhibition against thioredoxin reductase were found to induce apoptosis through activation of JNK and p38 MAPK pathways. In conclusion, this study had evaluated and identified enone-based bioactive compounds that display selective and dose-dependent inhibition of thioredoxin reductase activity in susceptible cell lines, which led to activation of p38 and JNK MAPK signaling pathways that at least in part triggered the apoptotic cascade. Taken together, the findings have indicated the potential of designing and developing these compounds that exert their anti-tumor effects through selective inhibition of thioredoxin reductase. Citation Format: Kamila K. Kaminska, Joanne Jia-An Low, Wan-Ying Chua, Esther Woon, Eng-Hui Chew. Evaluation of anti-tumor enone-based bioactive compounds as specific thioredoxin reductase inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4480. doi:10.1158/1538-7445.AM2015-4480

A Thiol–Ene Coupling Approach to Native Peptide Stapling and Macrocyclization

We report the discovery of a peptide stapling and macrocyclization method using thiol-ene reactions between two cysteine residues and an α,ω-diene in high yields. This new approach enabled us to selectively modify cysteine residues in native, unprotected peptides with a variety of stapling modifications for helix stabilization or general macrocyclization. We synthesized stapled Axin mimetic analogues and demonstrated increased alpha helicity upon peptide stapling. We then synthesized stapled p53 mimetic analogues using pure hydrocarbon linkers and demonstrated their abilities to block the p53-MDM2 interaction and selectively kill p53 wild-type colorectal carcinoma HCT-116 cells but not p53 null cells. In summary, we demonstrated a robust and versatile peptide stapling method that could be potentially applied to both synthetic and expressed peptides.

The different radiation response and radiation-induced bystander effects in colorectal carcinoma cells differing in p53 status

Radiation-induced bystander effect, appearing as different biological changes in cells that are not directly exposed to ionizing radiation but are under the influence of molecular signals secreted by irradiated neighbors, have recently attracted considerable interest due to their possible implication for radiotherapy. However, various cells present diverse radiosensitivity and bystander responses that depend, inter alia, on genetic status including TP53, the gene controlling the cell cycle, DNA repair and apoptosis. Here we compared the ionizing radiation and bystander responses of human colorectal carcinoma HCT116 cells with wild type or knockout TP53 using a transwell co-culture system. The viability of exposed to X-rays (0–8Gy) and bystander cells of both lines showed a roughly comparable decline with increasing dose. The frequency of micronuclei was also comparable at lower doses but at higher increased considerably, especially in bystander TP53-/- cells. Moreover, the TP53-/- cells showed a significantly elevated frequency of apoptosis, while TP53+/+ counterparts expressed high level of senescence. The cross-matched experiments where irradiated cells of one line were co-cultured with non-irradiated cells of opposite line show that both cell lines were also able to induce bystander effects in their counterparts, however different endpoints revealed with different strength. Potential mediators of bystander effects, IL-6 and IL-8, were also generated differently in both lines. The knockout cells secreted IL-6 at lower doses whereas wild type cells only at higher doses. Secretion of IL-8 by TP53−/− control cells was many times lower than that by TP53+/+ but increased significantly after irradiation. Transcription of the NFκBIA was induced in irradiated TP53+/+ mainly, but in bystanders a higher level was observed in TP53−/− cells, suggesting that TP53 is required for induction of NFκB pathway after irradiation but another mechanism of activation must operate in bystander cells.

Active human nucleolar organizer regions are interspersed with inactive rDNA repeats in normal and tumor cells.

The synthesis of rRNA is a key determinant of normal and malignant cell growth and subject to epigenetic regulation. Yet, the epigenomic features of rDNA arrays clustered in nucleolar organizer regions are largely unknown. We set out to explore for the first time how DNA methylation is distributed on individual rDNA arrays. Here we combined immunofluorescence detection of DNA modifications with fluorescence hybridization of single DNA fibers, metaphase immuno-FISH and methylation-sensitive restriction enzyme digestions followed by Southern blot. We found clustering of both hypomethylated and hypermethylated repeat units and hypermethylation of noncanonical rDNA in IMR90 fibroblasts and HCT116 colorectal carcinoma cells. Surprisingly, we also found transitions between hypo- and hypermethylated rDNA repeat clusters on single DNA fibers. Collectively, our analyses revealed co-existence of different epialleles on individual nucleolar organizer regions and showed that epi-combing is a valuable approach to analyze epigenomic patterns of repetitive DNA.

P-227 Cordycepin inhibits lipopolysaccharide-induced cell migration and invasion in human colorectal carcinoma HCT116 cells through down-regulation of prostaglandin E2 receptor EP4

P-227 Cordycepin inhibits lipopolysaccharide-induced cell migration and invasion in human colorectal carcinoma HCT116 cells through down-regulation of prostaglandin E2 receptor EP4