Colorectal Adenocarcinoma HT-29 Research Articles

Abstract 3613: Mononuclear phagocytic system occupancy to increase nanomedicines based treatment efficacy

Abstract Introduction: To efficiently deliver treatment, nanomedicines must exhibit sufficient blood bioavailability for further accumulation at the target site. So far, a large part of the administered dose remains useless due to the high rate of clearance by the mononuclear phagocytic system (mainly by Kupffer cells). Here we propose a new approach to redefine nanomedicines bioavailability by priming the body before receiving the treatment as a sequential administration of a nanoprimer before the nanomedicine. The nanoprimer is a nanoparticle designed to transiently occupy the main pathway responsible for the limited bioavailability of nanomedicines. As such, the nanoprimer allows to redefine the bioavailability of different nanomedicines and improve treatment’s outcomes for a large panel of therapeutic agents (e. g. small molecules or nucleic acids). Methods: Optimization of nanomedicines bioavailability was performed using a liposomal nanoprimer with specific physico-chemical properties. First we evaluated the nanoprimer’s biodistribution by in vivo imaging system, and its accumulation within the different hepatic cell populations by flow cytometry. Then we evaluated the impact of this nanoprimer on the bioavailability of different nanomedicines: for this, blood bioavailability and biodistribution of irinotecan loaded liposomes or mRNA loaded nanoparticles (lipidic and polymeric) were evaluated by HPLC and fluorescence respectively. We compared distribution of each nanomedicine administered intravenously alone or 10min after a single intravenous (IV) injection of nanoprimer on HT29 (colorectal adenocarcinoma) xenografted mice. Finally, the impact of nanoprimer on efficacy was evaluated by a tumor growth delay experiment for irinotecan loaded liposomes by IV administration of nanoprimer 10min before irinotecan loaded liposomes in HT29 xenografted mice. Treatment cycle was repeated one week later. For nucleic acid loaded nanoparticles, the impact of the nanoprimer was evaluated by measuring transfection efficiency on HT29 xenografted mice 24h after IV administration of the nanoprimer 10min before nanomedicine. Results: liposomal nanoprimers present a rapid accumulation in the liver with a preferential localization in Kupffer cells and liver sinusoidal endothelial cells. This accumulation leads to transient cells saturation and decreased hepatic trapping of the nanomedicines. Increased bioavailability resulted in a higher accumulation of irinotecan loaded liposomes within the HT29 tumor and in an increased efficacy. Such a bioavailability increase is also related with the transfection profile obtained for the nucleic acid loaded nanoparticles. Here we showed that a same nanoprimer could be used to prime the body to receive different types of nanomedicines and improve treatment’s outcomes. Such approach may decrease compromises between bioavailability, efficacy and toxicity. Citation Format: matthieu germain, Laurence Poul, Julie Devalliere, Marion Paolini, Audrey Darmon, Maxime Bergere, Oceane Jibault, Francis Mpambani. Mononuclear phagocytic system occupancy to increase nanomedicines based treatment efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3613.

Abstract 4423: Antitumor activity of the novel oral highly selective Wee1 inhibitor Debio 0123

Abstract Background: The Wee1 tyrosine kinase is activated upon DNA damage and regulates the G2-M cell cycle checkpoint. Inhibition of Wee1, in conjunction with additional genetic alterations and/or addition of a DNA damaging agent, results in mitotic catastrophe and apoptosis of cancer cells, offering an attractive approach to treating cancer. The aim of the present study was to characterize the pharmacological properties of the newly discovered, orally available, and highly selective Wee1 inhibitor Debio 0123. Methods: Profiling of Debio 0123 was performed on 465 selected kinases in a cell-free system. Effects of Debio 0123 on downstream signaling were analyzed by ELISA and western blot in HT29 (colorectal adenocarcinoma) and A427 (lung carcinoma) cell lines. The in vitro growth inhibition activity of Debio 0123 was defined in a broad number of human cancer cell lines after 72h using a proliferation monolayer assay. Debio 0123 in vivo anti-tumoral activity was assessed in an A427 subcutaneous xenograft model in athymic nude mice. Debio 0123 pharmacodynamic effects were also evaluated after oral dosing in surrogate tissues of mice, rats and monkeys by immunohistochemistry. Results: Debio 0123 is a highly selective ATP-competitive Wee1 inhibitor with an IC50 in the low nanomolar range. Debio 0123 inhibited Cdk1 (Cdc2) phosphorylation at Y15, a direct substrate of Wee1 in cells. Debio 0123 also induced dose- and time-dependent increased mitosis and unrepaired DNA damage shown by increased phosphorylation of histone H3 and γH2AX foci formation. In vitro activity was further studied in a large panel of cancer cell lines. Debio 0123 demonstrated submicromolar cytotoxic activity on multiple cell lines from various histotypes. In vivo, Debio 0123 was shown to inhibit phosphorylation of Cdk1 in tissues of multiple species and induction of DNA damage in a xenograft model. When administered orally once daily for 28 consecutive days, Debio 0123 induced dose-dependent anti-tumor activity and was well tolerated at all doses tested. At 30 mg/kg, treatment with Debio 0123 resulted in tumor regression. Conclusion: Altogether these results demonstrate that Debio 0123 is a highly selective and potent Wee1 inhibitor able to prevent Cdk1 phosphorylation in multiple species and to induce apoptosis through accumulation of unrepaired DNA damage both in vitro and in vivo that result in tumor regression. The advancement of Debio 0123 into clinical studies may provide improved therapeutic outcomes for patients with cancer. Citation Format: Colin O'Dowd, Gerald Gavory, Frank Burkamp, Adam Treder, Caroline Boyd, Tim Harrison, Frederic Massière, Astrid Glück, Christophe Chardonnens, Andrea Zaffalon, Stefania Rigotti, Robert Mader, Grégoire Vuagniaux, Anne Vaslin Chessex. Antitumor activity of the novel oral highly selective Wee1 inhibitor Debio 0123 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4423.

Genoprotective Effects of Essential Oil Compounds Against Oxidative and Methylated DNA Damage in Human Colon Cancer Cells.

Essential oils (EO) are widely used in foods as flavoring and preservative agents. Many of the biological activities of EO have been attributed to major essential oil compounds (EOC) but their direct interaction with colonic epithelial cells and their genotoxic and genoprotective effects are not well established. In this study, the cytotoxicity and genotoxicity of EOC including nerolidol, thymol, geraniol, methylisoeugenol, eugenol, linalool, and a commercial blend (Agolin) were determined. Furthermore, the genoprotective effects of EOC against oxidative and methylating damage were assessed using the comet assay in HT-29 colorectal adenocarcinoma cells. The majority of EOC were cytotoxic to HT-29 cells at or above 250 ppm after 24 hr exposure. At noncytotoxic doses, none of the EOC was genotoxic in the comet assay. Genoprotection against oxidative DNA damage was observed for nerolidol (at 62.5 ppm), thymol (at 12.5 ppm), geraniol, and methylisoeugenol (both at 125 ppm), as well as linalool and Agolin (both at 250 ppm). Thymol was the most protective compound against oxidative DNA damage and geraniol (at 125 ppm) also protected cells against methylating DNA damage. This study highlights the potential of EOC such as thymol to protect the colonic epithelium against oxidative DNA damage and geraniol against methylating DNA damage. Further in vivo studies are needed to confirm these findings for safety and efficacy to exploit their potential pharmaceutical or nutraceutical uses for colonic health.

Structures and Bioactivities of Six New Triterpene Glycosides, Psolusosides E, F, G, H, H1, and I and the Corrected Structure of Psolusoside B from the Sea Cucumber Psolus fabricii.

Seven sulfated triterpene glycosides, psolusosides B (1), E (2), F (3), G (4), H (5), H1 (6), and I (7), along with earlier known psolusoside A and colochiroside D have been isolated from the sea cucumber Psolus fabricii collected in the Sea of Okhotsk. Herein, the structure of psolusoside B (1), elucidated by us in 1989 as a monosulfated tetraoside, has been revised with application of modern NMR and particularly MS data and proved to be a disulfated tetraoside. The structures of other glycosides were elucidated by 2D NMR spectroscopy and HR-ESI mass-spectrometry. Psolusosides E (2), F (3), and G (4) contain holostane aglycones identical to each other and differ in their sugar compositions and the quantity and position of sulfate groups in linear tetrasaccharide carbohydrate moieties. Psolusosides H (5) and H1 (6) are characterized by an unusual sulfated trisaccharide carbohydrate moiety with the glucose as the second sugar unit. Psolusoside I (7) has an unprecedented branched tetrasaccharide disulfated carbohydrate moiety with the xylose unit in the second position of the chain. The cytotoxic activities of the compounds 2–7 against several mouse cell lines—ascite form of Ehrlich carcinoma, neuroblastoma Neuro 2A, normal epithelial JB-6 cells, and erythrocytes—were quite different, at that hemolytic effects of the tested compounds were higher than their cytotoxicity against other cells, especially against the ascites of Ehrlich carcinoma. Interestingly, psolusoside G (4) was not cytotoxic against normal JB-6 cells but demonstrated high activity against Neuro 2A cells. The cytotoxic activity against human colorectal adenocarcinoma HT-29 cells and the influence on the colony formation and growth of HT-29 cells of compounds 1–3, 5–7 and psolusoside A was checked. The highest inhibitory activities were demonstrated by psolusosides E (2) and F (3).

Bacteriophage Therapy Testing Against Shigella flexneri in a Novel Human Intestinal Organoid-Derived Infection Model.

Enteric bacterial pathogens cause diarrheal disease and mortality at significant rates throughout the world, particularly in children younger than 5 years. Our ability to combat bacterial pathogens has been hindered by antibiotic resistance, a lack of effective vaccines, and accurate models of infection. With the renewed interest in bacteriophage therapy, we sought to use a novel human intestinal model to investigate the efficacy of a newly isolated bacteriophage against Shigella flexneri. An S. flexneri 2457T-specific bacteriophage was isolated and assessed through kill curve experiments and infection assays with colorectal adenocarcinoma HT-29 cells and a novel human intestinal organoid-derived epithelial monolayer model. In our treatment protocol, organoids were generated from intestinal crypt stem cells, expanded in culture, and seeded onto transwells to establish 2-dimensional monolayers that differentiate into intestinal cells. The isolated bacteriophage efficiently killed S. flexneri 2457T, other S. flexneri strains, and a strain of 2457T harboring an antibiotic resistance cassette. Analyses with laboratory and commensal Escherichia coli strains demonstrated that the bacteriophage was specific to S. flexneri, as observed under co-culture conditions. Importantly, the bacteriophage prevented both S. flexneri 2457T epithelial cell adherence and invasion in both infection models. Bacteriophages offer feasible alternatives to antibiotics for eliminating enteric pathogens, confirmed here by the bacteriophage-targeted killing of S. flexneri. Furthermore, application of the organoid model has provided important insight into Shigella pathogenesis and bacteriophage-dependent intervention strategies. The screening platform described herein provides proof-of-concept analysis for the development of novel bacteriophage therapies to target antibiotic-resistant pathogens.

3D hybrid structures based on biomimetic membranes and Caryophyllus aromaticus - “green” synthesized nano-silver with improved bioperformances

The paper describes an innovative bio-design of some hybrid nanoarchitectures containing bioartificial membranes and silver nanoparticles phytogenerated by using a natural extract Caryophyllus aromaticus (cloves) that contains many bioactive compounds. Two kinds of liposomes with and without chlorophyll a (Chla) obtained through thin film hydration method were used to achieve bio-green-generated hybrids by a simple, cost effective bottom-up approach. The characteristic peaks of CE-nAg monitored by UV–Vis absorption have firstly demonstrated the biohybrids formation. The slightly blue shift and fluorescence quenching observed by fluorescence emission spectra highlighted the formation of hybrid systems by biointeraction between lipid vesicles and silver nanoparticles. The incorporation of silver nanoparticles in lipid vesicles resulted in significant changes of FT-IR spectra of liposomes, indicating a reorganization of biomimetic membranes. All the microscopic methods (SEM, AFM and TEM) confirmed the biosynthesis of “green” AgNPs together with associated biohybrids, their spherical and quasi-spherical shapes with nano-scaled size. By TEM assay it was shown that CE-nAg are surrounded by petal like cloud structures that consist of biopolymers like proteins or polysaccharides and other phytochemicals arising from clove extract. EDS spectra confirmed the formation of phyto-nanoAg and also the presence of silver in the biohybrids. In addition, Selected Area Electron Diffraction showed characteristic polycrystalline ring patterns for a cubic structure of the clove-generated AgNPs. The hybrid materials showed efficient physical stability,i.e. ξ value of −28.0 mV (for biohybrids without Chla, BH) and of −31.7 mV (for biohybrids labelled with Chla, Chla-BH), assured by strong electrostatic repulsive forces between particles. The “green” nano-silver particles (CE-nAg) showed remarkable antioxidant activity (AA = 90.2%). The biohybrids loaded with clove-AgNPs proved to be more effective, scavenging about 98.8% of free radicals (in case of Chla-BH), and of 92.6% (in case of BH). The antibacterial effectiveness showed that green AgNPs combine in a synergistic manner the antibacterial properties of clove extract with those of silver, resulting in an enhancement of inhibition diameter, by 20%. Chla-BH proved to be more potent against Escherichia coli, than BH, exhibiting an inhibition diameter of 42 mm. Regarding the in vitro cytotoxicity against tumour cells, the CE-nAg concentration significantly influenced the cell viability, i.e. IC50 was 3.6% (v/v) for HT-29 cells. Chla-BH was more effective against HT-29 cancer cells at the concentrations ranging from 0 to 18% (v/v), when the normal cells were not affected. Clove-generated AgNPs exhibited haemolytic activity against hRBCs, while the biohybrids were haemocompatible.The action mechanism on the two cell lines (mouse fibroblast L929 cells and human colorectal adenocarcinoma HT-29 cells) investigated by fluorescence microscopy demonstrated that CE-nAg killed almost all the cells (94%) through necrosis at a concentration of 33.4% (v/v). The treatment of HT-29 cells with BH resulted in: 71.5% viable cells, 19.5% apoptotic and only 9% necrotic cells, while in the case of Chla-BH treatment, only 77.5% cells were viable, 16% cells were apoptotic and 6.5% were necrotic. In this way, the developed silver-based nanoparticles can represent viable promoters to develop new biohybrids with improved features, e.g. antioxidant and antibacterial effectiveness, haemolytic activity and greater specificity towards tumour cells.

Участие α5-цепи ламининов в регуляции процесса дифференцировки клеток колоректального рака

The effect of endogenous expression of the laminin α5 chain on the phenotype of colorectal cancer cells has been studied. For this purpose, the knockdown of LAMA5 gene coding for the α5 laminin chain in the HT29 colorectal adenocarcinoma cell line was carried out. The data on gene expression at both mRNA and protein levels indicate that the α5 laminin chain is involved in the regulation of cell differentiation. For example, a decrease in the expression of such markers of differentiation of the intestinal epithelium as SI, KRT20, ANXA13 and MUC13 was observed. In addition, the expression of the intestinal epithelial stem cell marker LRG5 and CD44 transmembrane protein was increased. Changing the phenotype of HT29 cells may be a result of the effect of the laminin α5 chain on the activity of the Wnt signaling pathway. colorectal cancer, laminin, differentiation, dedifferentiation, intestinal epithelium stem cell marker, HT29, LAMA5, LRG5 The study was funded by the Russian Science Foundation (Project no. 17-14-01338).

Photo-induced eradication of human colorectal adenocarcinoma HT-29 cells by carbon monoxide (CO) delivery from a Mn-based green luminescent photoCORM

Exogenous CO delivery to cellular targets for salutary purposes can be readily achieved with photoactive metal carbonyl complexes (photoCORMs). Mn(I)-based photoCORMs are often favored for their ability to release CO upon triggering with visible light (suitable for phototherapy of cancer). To date, the reported Mn(I)-based photoCORMs are all non-luminescent and hence the entry of these CO donors cannot be tracked within the targeted sites. A new luminescent Mn(I) photoCORM namely [Mn(CO)3(phen)(Pipdansyl)](CF3SO3) (3) has been reported. Synthetic strategy to isolate photoCORM 3 and its previous analogue [Mn(CO)3(phen)(Imdansyl)](CF3SO3) (2) has been highlighted to establish protocols for the future isolation of luminescent Mn(I)-based photoCORMs that could be employed as trackable photoCORMs for CO delivery. The entrance of 3 into human colorectal adenocarcinoma HT-29 cells has been visualized by confocal microscopic studies. Eradication of the cancer cells by CO delivery from 3 under visible light illumination has also been demonstrated.

In vitro evaluation of antioxidant and anti-proliferative activities of Gypsophila sphaerocephala (Caryophyllaceae) extracts together with their phenolic profiles

This study was designed to investigate the antioxidant and antiproliferative activities of water and methanol extracts of endemic plant Gypsophila sphaerocephala subsp cappadocica in conjuction with phenolic profiles. Individual phenolics of the extracts were identified and quantified by RP-HPLC analysis. Antioxidant potentials of the extracts were evaluated by DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS [2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] radical scavenging capacity tests, cupric ion reducing antioxidant capacity (CUPRAC) method and Fe2+ chelating assay. Antiproliferative activities of the extracts were tested against MCF-7 (breast adenocarcinoma), HT-29 (colorectal adenocarcinoma) and HepG2 (hepatocellular carcinoma) cell lines. RP-HPLC analysis showed that methanol extract was richer than water extract in terms of phenolic content. In parallel to the phenolic contents, methanol extract showed higher antioxidant activity than water extract by DPPH, CUPRAC and Fe2+ chelating tests while water extract exhibited higher activity by ABTS method. Moreover, methanol extract displayed 1.8-fold, 4.3-fold and 2.6-fold more antiproliferative activity than water extract against MCF-7 cells, HepG2 cells and HT-29 cells, respectively. However, both extracts were found to show moderate antioxidant and antiproliferative activity compared to positive controls. These results suggest that Gypsophila sphaerocephala may be used as a promising source for food and nutraceutical industries due to its considerable antioxidant and antiproliferative potentials together with its rich phenolic content.

Cladolosides O, P, P1-P3 and R, triterpene glycosides with two novel types of carbohydrate chains from the sea cucumberCladolabes schmeltzii. Inhibition of cancer cells colony formation and its synergy with radioactive irradiation

The sea cucumber Cladolabes schmeltzii (Sclerodactylidae, Dendrochirotida) contains diverse glycosides, including several dozen individual compounds. Six new triterpene oligoglycosides, cladolosides O (1), P (2), P1 (3), P2 (4), P3 (5) and R (6), were isolated from this sea cucumber. Their structures were elucidated by 2D NMR spectroscopy and HR ESI mass spectrometry. Cladoloside O (1) has a pentasaccharide carbohydrate chain. Cladolosides of the group P and cladoloside R (6) include novel hexasaccharide carbohydrate chains with different positions of non-methylated terminal sugar units. All the isolated compounds demonstrate strong cytotoxic activities against cells of mouse Ehrlich carcinoma cells (ascite form) and mouse erythrocytes. The cytotoxicity of these compounds against human colorectal adenocarcinoma HT-29 cells was somewhat lower. The compounds 1–6 also inhibit the colony formation and growth of HT-29 cells at non-cytotoxic concentrations. The highest inhibition effect was demonstrated by cladoloside P1 (3). Moreover, synergism of effects of radioactive irradiation and non-toxic dose of compounds 1–6 on colony formation of HT-29 cells was observed. Cladolosides P2 (4) and P3 (5) were the most active and increased the inhibitory effect of radiation by more than 70%. The metabolic network demonstrating the biosynthetic pathways to carbohydrate chains of the glycosides of C. schmeltzii, based on a comparison of their structures, was constructed.

Cladolosides C4, D1, D2, M, M1, M2, N and Q, new triterpene glycosides with diverse carbohydrate chains from sea cucumber Cladolabes schmeltzii. An uncommon 20,21,22,23,24,25,26,27-okta-nor-lanostane aglycone. The synergism of inhibitory action of non-toxic dose of the glycosides and radioactive irradiation on colony formation of HT-29 cancer cells

Eight new triterpene olygoglycosides, cladolosides C4 (1), D1 (2), D2 (3), M (4), M1 (5), M2 (6), N (7) and Q (8), were isolated from the tropical Indo-West Pacific sea cucumber Cladolabes schmeltzii (Sclerodactylidae, Dendrochirotida). Structures of these glycosides were elucidated by 2D NMR spectroscopy and HR ESI mass spectrometry. A novel hexasaccharide carbohydrate chain having xylose residues as the first, second and third sugars was found in the glycoside 7. Cladoloside C4 (1) contains an uncommon 20,21,22,23,24,25,26,27-octa-norlanostane aglycone. Cladolosides D1 (2), D2 (3) and Q (8) were new representatives of the hexaosides with a non-methylated terminal sugar unit in the “upper” half-chain. Cytotoxic activities of the isolated compounds against ascite form of mouse Ehrlich carcinoma cells, mouse erythrocytes and human colorectal adenocarcinoma HT-29 cells were examined and their structure-activity relationships were analyzed. In addition, the majority of tested compounds, except for cladoloside D2 (3), inhibited the colony formation and growth of HT-29 cells at non-cytotoxic concentrations. The highest inhibitory activity was demonstrated by cladoloside M1 (5). Moreover, synergism of effects of radioactive irradiation and non-toxic dose of compounds 1–8 decreasing the number of colonies of HT-29 cells was observed. Cladoloside N (7) was the most active and increased the inhibitory effect from radiation by 75%. The biosynthetic transformations of the aglycones are discussed.

PO-036 Traditional chinese medicine Ze-Qi-TANG formula induces apoptosis and S phase arrest via ROS-dependent JNK and ERK activation in lung cancer

IntroductionDespite being significant advances in colon cancer treatments, recurrence and chemoresistance remain an important challenge in the treatment of patients. During the process of autophagy, cancer cells acquire anoikis resistance and escape chemotherapy. The High Mobility Group Box 1 (HMGB1) molecule is a key mediator of autophagy and can be exploited to develop effective targeted anticancer therapies. Glycyrrhizin and quercetin are natural inhibitor of high mobility box 1 protein (HMGB1).We studied the anticancer activity of glycyrrhizic acid and quercetin in SW480, HT29 and DLD1 colorectal adenocarcinoma cells using alamar blue and trypan blue exclusion assay and determined their IC50. We also proposed to illustrate the anticancer mechanism of these natural compounds by studying cell cycle response, proteome profile and response of cell cycle specific genes in HT29 and DLD1 colorectal cancer cells. natural inhibitor of high mobility box 1 protein (HMGB1).We studied the anticancer activity of glycyrrhizic acid and quercetin in SW480, HT29 and DLD1 colorectal adenocarcinoma cells using alamar blue and trypan blue exclusion assay and determined their IC50. We also proposed to illustrate the anticancer mechanism of these natural compounds by studying cell cycle response, proteome profile and response of cell cycle specific genes in HT29 and DLD1 colorectal cancer cells.Material and methodsCell cycle analysis was performed using a propidium iodide based staining assay in a Muse flow cell analyzer. For proteomics response, R and D proteome-profiler-antibody-arrays for oncology panel for 84 oncoproteins were assayed. A SYBR green based approach for the quantification of selected genes was considered in qPCR analysis.Results and discussionsBoth glycyrrhizic acid and quercetin showed altered protein and gene expression in HT29 and DLD colorectal adenocarcinoma cells.ConclusionAs evident from their low IC50s (80 and 160mcg/ml) against the colorectal cells, both quercetin and glycyrrhizic acid showed a promising scaffold for medicinal chemistry to advance anticancer drug discovery.

PO-033 Anticancer activity of natural high mobility group BOX1 inhibitors in colorectal cancer cells

IntroductionDespite being significant advances in colon cancer treatments, recurrence and chemoresistance remain an important challenge in the treatment of patients. During the process of autophagy, cancer cells acquire anoikis resistance and escape chemotherapy. The High Mobility Group Box 1 (HMGB1) molecule is a key mediator of autophagy and can be exploited to develop effective targeted anticancer therapies. Glycyrrhizin and quercetin are natural inhibitor of high mobility box 1 protein (HMGB1).We studied the anticancer activity of glycyrrhizic acid and quercetin in SW480, HT29 and DLD1 colorectal adenocarcinoma cells using alamar blue and trypan blue exclusion assay and determined their IC50. We also proposed to illustrate the anticancer mechanism of these natural compounds by studying cell cycle response, proteome profile and response of cell cycle specific genes in HT29 and DLD1 colorectal cancer cells. natural inhibitor of high mobility box 1 protein (HMGB1).We studied the anticancer activity of glycyrrhizic acid and quercetin in SW480, HT29 and DLD1 colorectal adenocarcinoma cells using alamar blue and trypan blue exclusion assay and determined their IC50. We also proposed to illustrate the anticancer mechanism of these natural compounds by studying cell cycle response, proteome profile and response of cell cycle specific genes in HT29 and DLD1 colorectal cancer cells.Material and methodsCell cycle analysis was performed using a propidium iodide based staining assay in a Muse flow cell analyzer. For proteomics response, R and D proteome-profiler-antibody-arrays for oncology panel for 84 oncoproteins were assayed. A SYBR green based approach for the quantification of selected genes was considered in qPCR analysis.Results and discussionsBoth glycyrrhizic acid and quercetin showed altered protein and gene expression in HT29 and DLD colorectal adenocarcinoma cells.ConclusionAs evident from their low IC50s (80 and 160mcg/ml) against the colorectal cells, both quercetin and glycyrrhizic acid showed a promising scaffold for medicinal chemistry to advance anticancer drug discovery.

Abstract LB-072: Redefine nanomedicine products bioavailability to improve anti-tumor efficacy

Abstract Introduction: The benefit of a nanomedicine is due to its bioavailability, its intrinsic efficacy balanced with its toxicity profile. The nanomedicine should exhibit sufficient blood bioavailability for efficient accumulation at the target site. So far, a large part of the administered dose remains useless due to the high rate of clearance by the mononuclear phagocytic system (mainly by hepatic Kupffer cells). Enhanced bioavailability can be achieved by modifying physico-chemical properties of the nanomedicine but such modification affects also its efficacy and toxicity profiles. Ultimately, nanomedicines design results usually in a compromise between bioavailability, efficacy or toxicity. Here we propose a new approach to change the way nanomedicine are biodistributed, by priming the body to receive the treatment. This approach relies on the sequential administration of a nanoprimer before the nanomedicine. The nanoprimer is a nanoparticle designed to transiently occupy the main pathway responsible for the limited bioavailability of the nanomedicine. As such, the nanoprimer allows to redefine the bioavailability of the nanomedicine. Methods: For proof of concept we designed a liposomal nanoprimer with specific physico-chemical properties. Its biodistribution was evaluated by labelling with gold nanoparticles. 24h after intravenous (IV) administration on mice, gold quantification was performed on sampled organs by ICPMS. Toxicity evaluation was performed by 3 IV injections of nanoprimer spaced of 24h on mice followed by a one week follow up of body weight and clinical signs. Histological liver and spleen observations and transaminases titration were performed 1 and 7 days after last injection. Evaluation of the impact of nanoprimer on nanomedicines bioavailability was performed by in vivo follow up of a fluorescent nanomedicine IV injected 10min after nanoprimer IV injection. Tumor growth delay experiment was performed by IV administration of nanoprimer 10min before irinotecan loaded liposomes on mice xenografted with HT29 (colorectal adenocarcinoma) tumor model once the tumor reached 150mm3. Treatment was repeated one week later. Results: The liposomal nanoprimer presents a preferential hepatic accumulation. No signs of systemic or hepatic toxicity were observed with maximized dose of this nanoprimer. Nanomedicine bioavailability studies showed a transient increase of nanomedicine blood bioavailability correlated with a lower hepatic accumulation when combined to nanoprimer. Finally efficacy study showed that nanoprimer markedly enhanced anti-tumor efficacy of irinotecan loaded liposomes in the HT-29 model when compared to the nanomedicine alone. Body priming may benefit to a wide variety of existing products modulating their bioavailability and may open perspective to design new nanomedicines by decreasing the notion of compromise between bioavailability, efficacy and toxicity. Citation Format: Matthieu Germain, Laurence Poul, Marie-Edith Meyre, Marion Paolini, Francis Mpambani, Maxime Bergere, Agnes Pottier, Laurent Levy. Redefine nanomedicine products bioavailability to improve anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-072.

Butyrate Inhibits Indices of Colorectal Carcinogenesis via Enhancing α-Ketoglutarate-Dependent DNA Demethylation of Mismatch Repair Genes.

Butyrate, the fermentation end product of gut microbiota in the colon, is known for its antitumor effects, but the mechanisms remained to be defined. α-ketoglutarate (α-KG) mediates DNA demethylation and aberrant epigenetic modifications are associated with carcinogenesis. The objectives of this study are to evaluate the effects of butyrate on α-KG mediated epigenetic modification in colorectal adenocarcinoma HT-29 and Caco-2 cells. Butyrate suppressed proliferation, potentiated differentiation, and induced apoptosis in both HT-29 and Caco-2 cells, associated with enhanced expression of isocitrate dehydrogenase 1 (IDH1) and pyruvate dehydrogenase. Furthermore, butyrate upregulated acetyl-CoA and α-KG, concomitant with enhanced histone acetylation and DNA demethylation in the promoter of DNA mismatch repair (MMR) gene. Knocking down IDH1 abolished the positive effects of butyrate on CRC apoptosis and MMR protein expression, in conjunction with reduced α-KG content. Importantly, α-KG supplementation recovered the beneficial effects of butyrate in IDH1-deficient cells. In summary, butyrate inhibits indices of colorectal carcinogenesis in an α-KG-dependent manner.

Krill oil, vitamin D and Lactobacillus reuteri cooperate to reduce gut inflammation.

Current research into original therapies to treat intestinal inflammation is focusing on no-drug therapies. KLD is a mixture of krill oil (KO), probiotic Lactobacillus reuteri (LR), and vitamin D (VitD3). The aim of this study was to assess in vitro and in vivo the potential cooperative effects of KLD in reducing gut inflammation. Colorectal adenocarcinoma cell lines, CACO2 and HT29, and C57BL/6 mice were used for in vitro and in vivo analyses, respectively. Cells were exposed to cytomix (interferon gamma + tumour necrosis factor alpha (TNF-α)) to induce inflammation or co-exposed to cytomix and KO, LR and VitD3 alone or to cytomix and KLD. Animals were treated for 7 days with dextran sodium sulphate (DSS) to induce colitis or with DSS and KLD. In vitro assays: F-actin expression was analysed by immunofluorescence; scratch test and trans-epithelial electric resistance test were performed to measure wound healing; adhesion/invasion assays of adhesive and invasive Escherichia coli (AIEC) bacteria were made; mRNA expression of TNF-α, interleukin (IL)-8 and vitamin D receptor (VDR) was detected by quantitative PCR. In vivo assays: body weight, clinical score, histological score and large intestine weight and length were estimated; mRNA expression of TNF-α, IL-1β, IL-6, IL-10 by quantitative PCR; VDR expression was detected by quantitative PCR and immunohistochemistry. In vitro: KLD restores epithelial cell-cell adhesion and mucosal healing during inflammation, while decreases the adhesiveness and invasiveness of AIEC bacteria and TNF-α and IL-8 mRNA expression and increases VDR expression. In vivo: KLD significantly improves body weight, clinical score, histological score and large intestine length of mice with DSS-induced colitis and reduces TNF-α, IL-1β and IL-6 mRNA levels, while increases IL-10 mRNA and VDR levels. KLD has significant effects on the intestinal mucosa, strongly decreasing inflammation, increasing epithelial restitution and reducing pathogenicity of harmful commensal bacteria.

Structure‐Activity Analysis of Bisphenol‐Z Derivatives for Anticancer Activity

Much of the research surrounding bisphenol derivatives focuses on their use as polymers and plasticizers for industry. Research has demonstrated that bisphenol‐A (BPA) adversely affects humans via endocrine disruption through hormonal modulation. Despite the recent explosion in BPA research, little effort has been devoted to researching the biological activities of other bisphenol derivatives, including bisphenol‐Z (BPZ). Initial screening of BPZ revealed moderate in vitro anticancer activity against four diverse human cancer cell lines (A375 malignant melanoma, A‐172 glioblastoma, HT‐29 colorectal adenocarcinoma, and MCF7 breast adenocarcinoma) with IC50 values of approximately 65 μM following 24 hour treatments. In an attempt to enhance the activity of BPZ, seven analogs with varying degrees of polarity, lipophilicity, and sterics on the cyclohexane ring were synthesized. Screening of these analogs revealed that polar substitutions resulted in a loss of activity, while a simple methyl substitution had similar activity to BPZ. Anticancer activity was enhanced relative to BPZ following substitution with an ethyl, isopropyl, or N‐palmitoyl group at the 4‐ position. The most active compounds from this structure‐activity study were the N‐palmitoyl (LS4) and isopropyl (LS6) derivatives with 24 hour IC50 values ranging from 25 to 53 μM and 33 to 42 μM respectively.Given the increased activity of LS4 and LS6, additional studies were performed including a trypan blue exclusion assay and a Caspase‐Glo® 3/7 assay. The results of these studies revealed exclusion of trypan blue at supra‐IC50 concentrations six hours after treatments, suggestive of a non‐necrotic mechanism of death. To determine if caspase‐dependent apoptosis was involved, we performed a Caspase‐Glo® 3/7 assay. Functional screening of executioner caspase‐3 and ‐7 revealed significant increases in activity following a 12 hour treatment with LS4 in all cells lines. This effect was least evident in the MCF7 cells, potentially due to a mutation wherein these cells lack expression of caspase‐3. In contrast, LS6 failed to activate executioner caspases‐3 and ‐7 in these same cell lines.In conclusion, this structure‐activity study revealed that nonpolar substitution at the 4‐ position of BPZ enhanced anticancer activity. Additionally, membrane integrity remained intact following exposure to LS4 and LS6. While LS4 activated executioner caspases‐3/7, LS6 failed to activate this hallmark pathway of apoptotic death. Further investigations are ongoing to elucidate the mechanisms of cell death, identify putative cellular targets, and determine potential selectivity against normal human cell lines.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Papain Loaded Solid Lipid Nanoparticles for Colorectal Cancer Therapy

Background: Colorectal cancer (CRC) also known as bowl cancer is still one of the leading causes of cancer related mortality worldwide. Generally tumor cells are protecting themselves by fibrin coat and it is resistant to fibrinolytic degradation. Such a coated tumor appears as ‘self' to the immune system, and thus is not detected as a tumor by the immune system (i.e. natural killer cells). Hence, a potent proteolytic enzyme has to propose/ identify to dissolve the protective fibrin layer, exposing the tumor cell surface to chemotherapy and immune attack. In this research papain was considered to be the potential proteolytic agent, can break down the fibrin coat of cancer cell wall and ultimately the cancer cells are exposed to immune attack and help against the cancer. Secondly, the cytotoxic compound(s) directly deliver to cancer site without harm to normal cells. Methods: The attempt made to attain this objective, we were designed to fabricate the Papain loaded solid lipid nanoparticles (SLN) by melt dispersion-ultrasonication technique, and investigate the various formulation parameters. The papain loaded SLN was characterized by particle size analysis, zeta potential analysis, differential scanning calorimetry (DSC), Scanning electron microscopy (SEM), drug encapsulation efficiency, in vitro drug release, and in vitro cytotoxicity studies on HT-29 colorectal cancer cells. Results: The successful outcome of this research was that, the cetyl alcohol based SLN of papain were successfully prepared by using melt dispersion- ultrasonication method with maximum encapsulation efficiency with desired particle size. The release profile of the produced SLN was investigated in phosphate buffer media, and it showed prolonged release during 24 h. The drug release behaviour from the SLNs exhibited a biphasic pattern with the burst release at the initial stage and sustained release subsequently. Future perspective of this research is that need to investigate the SLN for the lymphatic uptake to produce local action on metastatic colorectal cancer. In vitro cell viability for P-SLNs was tested on colorectal Adenocarcinoma HT-29 cells by MTT assay. The results revealed that the in vitro cell viability against different concentrations of papain was 99% and 31 % which were treated with 5µg/ml and 80µg/ml concentration respectively. The cell viability of HT-29 cells was decreased signicantly from 94 % to 17 % treated with P-SLN at 5 µg/ml and 80 µg/ml concentration respectively. Therefore, the papain loaded SLNs exhibits a better performance on getting lower cell viability (or) equivalent high cytotoxicity than that of pure papain. Therefore, the SLNs delivery system plays a significant role in enhancing the cytotoxic efficacy of papain enzyme against colorectal cancer cells. Conclusion: In this research, we successfully formulated the P-SLN for the treatment of colorectal cancer therapy. We investigate the various formulation parameters, drug release profile and cytotoxic efficacy of P-SLN. Upon successful completion of this work, P-SLNs delivery system is predictable to produce enhanced cytotoxic efficacy against HT-29 colorectal cancer cells. The papain loaded SLNs can potentially be utilized as a drug delivery system for the treatment of colorectal cancer. In future, we will explore further to investigate the mechanism of anticancer activity and clinical investigation of P-SLN. Keywords: Papain, cytotoxic potential, solid lipid nanoparticles, cell viability, proteolytic enzyme, Colorectal cancer (CRC).

Anti-Proliferative and Apoptotic Effects of Polygonum cuspidatum and its Bioactive Compound, Emodin, in Colorectal Carcinoma HT-29 cells

Antiproliferative and apoptotic effects of ethyl acetate from the root of Polygonum cuspidatum (EAPC) and its bioactive compound, emodin, in colorectal adenocarcinoma HT-29 cells were investigated. HPLC analysis of major compounds, cell viability by MTT assay, the Comet assay, determination of reactive oxygen species (ROS) levels, apoptosis assay by flow cytometry and Western blot analysis were conducted. The results show significant antiproliferative effects of EAPC, IC50 = 141.4 μg/mL, and emodin isolated from EAPC, IC50 = 73.0 μg/mL, on HT-29 cells. HT-29 cells which were exposed to EAPC and emodin exerted apoptosis, after annexin V FITC/PI staining. EAPC (at 200 μg/mL) and emodin (at 100 μg/mL) increase DNA damage in HT-29 cells by 75.6% and 67.8%, respectively, supporting that apoptosis occurred when the cells were treated with EAPC and emodin. Cells treated with EAPC and emodin generated intracellular ROS. In addition, emodin at tested doses of 25-100 μg/mL suppressed nuclear factor κB (NFκB) and enhanced activator protein 1 (AP-1), which are involved in both proliferation and apoptosis events. EAPC and emodin demonstrated significant anti-proliferation of HT-29 cells through their pro-oxidant activity and contribution to oxidative stress, which in turn leads to cell death. EPAC and emodin inhibited HT-29 cell growth via apoptosis and could be considered as potential candidates for anti-colorectal cancer treatment.

Rubus sanctus Schreb. root extract alters the MicroRNA expression and inhibits tumor activities of colorectal cancer cell lines

Background: Colorectal cancer (CRC) is one of the most common cancers in the world. Although surgical and screening techniques have vastly improved in the last 30 years, chemotherapeutics have not advanced sufficiently for successful treatment. Objective: The aim of this study is to investigate the microRNA (miRNA) expression changes and anticancer agent potential of Rubus Sanctus Schreb. root extract (RRE) on LoVo and HT-29 colorectal adenocarcinoma cell lines. Materials and Methods: LoVo and HT-29 CRC cell lines treated with different concentrations of RRE to find growth inhibitory effect with WST-1 assay. Fifty percent growth inhibition and 25% growth inhibition concentrations further evaluated with annexin V, total caspase, cell cycle, and migration assays. Real-time polymerase chain reaction was used to investigate the expression differences in miRNA after extract treatment. Results: Cell proliferation was reduced 77.98% in HT-29 cells after RRE treatment ( P P P P P Conclusion: Our data show for the first time that RRE has the capability to inhibit CRC cell proliferation and invasion and alter epigenetic mechanisms. Although further studies should be conducted on this topic, RRE is thought to be a potential candidate for the future studies regarding new therapy options. Abbreviation used: RRE: Rubus Sanctus Schreb. root extract; CRC: Colorectal cancer; GI 50 , GI 25: 50% and 25% growth inhibition; miRNA: MicroRNA; MGMT: O-6-Methylguanine-DNA methyltransferase; UTR: Untranslated region; Akt: Protein kinase B; PI3K: Phosphoinositide 3-kinase; NF-κB: Nuclear factor kappaB; iNOS: Inducible nitric oxide synthase; COX-2: cyclooxygenase 2; TNFα: tumor necrosis factor alpha; IL-6: Interleukin 6; DMEM: Dulbecco's Modified Eagle's medium; RPMI-1640: Roswell Park Memorial Institute 1640 medium; HPLC: High-performance liquid chromatography; UV-Vis: Ultraviolet-visible; FBS: Fetal bovine serum; OD: Optical density; RFU: Relative fluorescence unit; PE: Phycoerythrin; 7-AAD: 7-Aminoactinomycin D; PBS: Phosphate-buffered saline; EGFR: Epidermal growth factor receptor; 5-FU: Fluorouracil; MSI: Microsatellite instability; NSCLC: Nonsmall cell lung cancer; TGF-β: Transforming growth factor beta; EMT: Epithelial–mesenchymal transition