High-Fructose Diet Alters Intestinal Microbial Profile and Correlates with Early Tumorigenesis in a Mouse Model of Barrett’s Esophagus

Esophageal adenocarcinoma (EAC) is mostly prevalent in industrialized countries and has been associated with obesity, commonly linked with a diet rich in fat and refined sugars containing high fructose concentrations. In meta-organisms, dietary components are digested and metabolized by the host and its gut microbiota. Fructose has been shown to induce proliferation and cell growth in pancreas and colon cancer cell lines and also alter the gut microbiota. In a previous study with the L2-IL-1B mouse model, we showed that a high-fat diet (HFD) accelerated EAC progression from its precursor lesion Barrett’s esophagus (BE) through changes in the gut microbiota. Aiming to investigate whether a high-fructose diet (HFrD) also alters the gut microbiota and favors EAC carcinogenesis, we assessed the effects of HFrD on the phenotype and intestinal microbial communities of L2-IL1B mice. Results showed a moderate acceleration in histologic disease progression, a mild effect on the systemic inflammatory response, metabolic changes in the host, and a shift in the composition, metabolism, and functionality of intestinal microbial communities. We conclude that HFrD alters the overall balance of the gut microbiota and induces an acceleration in EAC progression in a less pronounced manner than HFD.

Peroxiporins Are Induced Upon Oxidative Stress Insult and Are Associated with Oxidative Stress Resistance in Colon Cancer Cell Lines

Oxidative stress can induce genetic instability and change cellular processes, resulting in colorectal cancer. Additionally, adaptation of oxidative defense causes therapy resistance, a major obstacle in successful cancer treatment. Peroxiporins are aquaporin membrane channels that facilitate H2O2 membrane permeation, crucial for regulating cell proliferation and antioxidative defense. Here, we investigated four colon cancer cell lines (Caco-2, HT-29, SW620, and HCT 116) for their sensitivity to H2O2, cellular antioxidative status, and ROS intracellular accumulation after H2O2 treatment. The expression of peroxiporins AQP1, AQP3, and AQP5 and levels of NRF2, the antioxidant transcription factor, and PPARγ, a transcription factor that regulates lipid metabolism, were evaluated before and after oxidative insult. Of the four tested cell lines, HT-29 was the most resistant and showed the highest expression of all tested peroxiporins and the lowest levels of intracellular ROS, without differences in GSH levels, catalase activity, nor NF2 and PPARγ levels. Caco-2 shows high expression of AQP3 and similar resistance as HT-29. These results imply that oxidative stress resistance can be obtained by several mechanisms other than the antioxidant defense system. Regulation of intracellular ROS through modulation of peroxiporin expression may represent an additional strategy to target the therapy resistance of cancer cells.

725 Immunomodulation by targeting PDL-1 in colon cancer using nuclear receptor 4A1 (NR4A1) antagonists

BackgroundThe nuclear orphan receptor 4A1 (NR4A1, Nur77, TR3) is overexpressed in multiple solid tumors including colorectal tumors and is a negative prognostic factor for patient survival.1–3 NR4A1 is expressed in colon cancer cells and exhibit pro-oncogenic activity4 and results of examination of several colon cancer cell lines show that PD-L1 expression is limited and NR4A1 and PD-L1 are co-expressed in SW480 and RKO colon cancer cell lines. Previous studies showed that PD-L1 was regulated by NR4A1 which activates transcription factor Sp1 bound to the PD-L1 gene promoter.5–7 Knockdown of NR4A1 or Sp1 by RNA interference or treatment with mithramycin an inhibitor of Sp-mediated transcription decreased expression of PD-L1 in RKO and SW480 colon cancer cell lines.MethodsSW480, RKO and MC-38 cells were used in this study. Cells were treated for 24 hrs with DIM series of compounds.ResultsCurrent data coupled with ongoing gene expression and PD-L1 promoter studies demonstrate that PD-L1 expression is regulated by NR4A1/Sp1 in colon cancer cells (figures 1–3). Bis-indole derived NR4A1 ligand that act as receptor antagonists have been developed in this laboratory and these compounds block pro-oncogenic NR4A1-regulated genes/pathways. Treatment of RKO and SW480 colon cancer cell lines with a series of potent 1,1-bis(3′-indolyl)-1-(3,5-disubstitutedphenyl) analogs decreased expression of PD-L1. These results show that bis-indole derived NR4A1 antagonists act as small molecule mimics of immunotherapeutics that target PD-L1. In vivo applications of NR4A1 ligands that target PD-L1 and their effects on tumor growth and immune surveillance are currently being investigated.ConclusionsBis-indole derived NR4A1 antagonists inhibit PD-L1 expression. NR4A1/SP1 regulates PD-L1 and is inhibited by NR4A1 antagonist. NR4A1 ligands such as DIM-3-Br-5-OCF3 were among the most potent of the substituted DIM compounds and ongoing in vivo studies show that this DIM compound also inhibits tumor growth in a syngenic mouse model (data not shown). Data from this study demonstrate the pro-oncogenic activity of NR4A1 and show that the synthetic buttressed analog DIM-3-Br-5-OCF3 acts as an NR4A1 antagonist and inhibits PD-L1 expression. These drugs can be developed for future clinical applications.Referenceswww.cancer.org/cancer/colon-rectal-cancer/about/key-statistics.html.Garcia-Villatoro et al., Effects of high-fat diet and intestinal aryl hydrocarbon receptor deletion on colon carcinogenesis. Am J Physiol Gastrointest Liver Physiol 2020;318(3):G451–G463.Safe S, Jin UH, Hedrick E, et al. Minireview: role of orphan nuclear receptors in cancer and potential as drug targets. Mol Endocrinol 2014;28(2):157–72.Maxwell MA, Muscat GE. The NR4A subgroup: immediate early response genes with pleiotropic physiological roles. Nucl Recept Signal 2006;4:e002.Lee SO, Li X, Hedrick E, et al. Diindolylmethane analogs bind NR4A1 and are NR4A1 antagonists in colon cancer cells. Mol Endocrinol 2014;28(10):1729–39.Safe S, Kim K. Non-classical genomic estrogen receptor (ER)/specificity protein and ER/activating protein-1 signaling pathways. J Mol Endocrinol 2008;41(5):263–75.Tao LH, Zhou XR, Li FC, Chen Q, Meng FY, Mao Y, et al. A polymorphism in the promoter region of PD-L1 serves as a binding-site for SP1 and is associated with PD-L1 overexpression and increased occurrence of gastric cancer. Cancer Immunol Immunother 2017;66(3):309–18.Abstract 725 Figure 1NR4A1 inactivation inhibits PD-L1 expression. SW480, RKO and MC-38 cells were transfected with siCtrl (non-specific oligonucleotide) and two oligonucleotides targeting NR4A1 (siNR4A1(1) and siNR4A1(2)) or PD-L1 (siPD-L1(1) and siPD-L1(2)) for 72 hrss. Protein expression from whole cell lysates were analyzed by western blots and effects on PD-L1 expression were determinedAbstract 725 Figure 2Sp1 inactivation inhibits PD-L1 expression. SW480, RKO and MC-38 cells were transfected with siCtrl and oligonucleotides targeting Sp1 (siSp1(1) and siSp1(2)) for 72 hrs as well as treated with Mithrsamycin (150 and 300 nM) for 24 hrs. Protein expression from was analyzed by western blots and effects on PD-L1 levels were determined.Abstract 725 Figure 3Role of NR4A1/Sp in regulation of PD-L1. SW480, RKO and MC-38 cells were treated with DIM-3-Br-5-OCF3 for 24 hrss and protein interactions with the GC-rich PD-L1 promoter region were analyzed by ChIP using primers encompassing GC-rich region of the promoter

G-749 Promotes Receptor Tyrosine Kinase TYRO3 Degradation and Induces Apoptosis in Both Colon Cancer Cell Lines and Xenograft Mouse Models

G-749 is an FLT3 kinase inhibitor that was originally developed as a treatment for acute myeloid leukemia. Some FLT3 kinase inhibitors are dual kinase inhibitors that inhibit the TAM (Tyro3, Axl, Mer) receptor tyrosine kinase family and are used to treat solid cancers such as non-small cell lung cancer (NSCLC) and triple-negative breast cancer (TNBC). AXL promotes metastasis, suppression of immune response, and drug resistance in NSCLC and TNBC. G-749, a potential TAM receptor tyrosine kinase inhibitor, and its derivative SKI-G-801, effectively inhibits the phosphorylation of AXL at nanomolar concentration (IC50 = 20 nM). This study aimed to investigate the anticancer effects of G-749 targeting the TAM receptor tyrosine kinase in colon cancer. Here, we demonstrate the potential of G-749 to effectively inhibit tumorigenesis by degrading TYRO3 via regulated intramembrane proteolysis both in vitro and in vivo. In addition, we demonstrated that G-749 inhibits the signaling pathway associated with cell proliferation in colon cancer cell lines HCT15 and SW620, as well as tumor xenograft mouse models. We propose G-749 as a new therapeutic agent for the treatment of colon cancer caused by abnormal TYRO3 expression or activity.

Goat’s Milk as a Potential Anti-proliferative against Colon Cancer Cell Lines

Aim: To investigate anti-proliferative effect of fresh and pasteurized goat milk against colon cancer cell lines (HT-29, HCT-116, CT-26). Study Design: Experimental study. Place and Duration of Study: Central Laboratory, Tissue Culture Laboratory, Universiti Sultan Zainal Abidin, Terengganu between January 2020 and April 2020. Methodology: Samples comprised of two types goat milk, which were fresh and pasteurized in powder form. The samples were analysed for the anti-proliferative effect by MTT assay, and IC50 value was determined. Then, cell apoptotic changes were observed by light inverted microscope by 24, 48 and 72 hours. Results: Experimental data showed that the fresh sample produce the highest yield (9.40%) than the pasteurized sample (7.17%). The fresh sample yielded the most potent cytotoxic value (0.28 ± 0.03), followed by pasteurized sample with value IC50 0.32 ± 0.02 against HCT-116 cells. Then, the anti-proliferative effect was observed on cell apoptotic changes by reduction of cell volume, cell densed, and presence of fragmentation and apoptotic bodies at 24, 48 and 72 hours treatment. Conclusion: In conclusion, the fresh sample of goat milk yielded the potent anti-proliferative effect than pasteurized sample.

The Role of Perforin

Some literature reviews have been carried out about the role of perforin in medicine. The first step involved a systematic search to identify relevant studies published between 2001 and 2019 in the following electronic databases - EBSCO host, Scopus, Science Direct, Web of Science, and Elsevier. By analyzing the available literature, it can be concluded that perforin plays an important role in cytoxical activity of natural killer cells (NK) and CD8+ T cell. NK and CD8+use the same mechanism for destroying target cells. This article cites the disease hemophagocytic lymphohistiocytosis (HLH) which is characterized by heavy abnormalities in the immune system. The point is that this disease is caused by perforin gene mutation. The key is the application of properly sensitized dendritic cells (DCs) because they are effective in immunotherapy against cancer. It may be effective in γ-irradiated colon cancer cell lines HT-29. Growth hormone inhibiting hormone (GIH) induces maturation and activation of DCs. In that way, GIH-Dcs shows increased cytotoxic activity and higher perforin and granzyme expression. So, this means that theoretical research has shown that efficient activity against cancer is induced when DCs are sensitized with γ-irradiated cancer cells. In that way, through a direct increase of cytotoxicity and indirect T cell activation,there can beanti-tumor activity. It is suggested to continue scientific research about the role of perforin in the future.

Riceberry rice bran protein hydrolyzed fractions induced apoptosis, senescence and G1/S cell cycle arrest in human colon cancer cell lines

Riceberry rice bran is the part of rice that has been scrubbed out during coloring process. There are various health benefits with high protein content and antioxidant ability. The hydrolyzed rice bran consists of diverse peptides that provide various bioactive properties. This work aimed to study the effect of hydrolyzed riceberry rice bran extracted on colon cancer cell lines (HT- 29 and SW- 620) compared to normal cell (PCS- 291- 010). The MTT assay result showed that our extract has less cytotoxicity on normal cell (PCS-291-010, IC50 = 6,680.00 µg/ml) compared to the colon cancer cell lines and has more effect on metastatic cancer cell line (SW-620, IC50 = 5,492.31 µg /ml) than non-metastatic cancer cell line (HT-29, IC50 =6,040.76 µg/ml). According to the DNA fragmentation pattern analysis, the ladder pattern indicated that the rice bran extract can induce the apoptosis process in SW-620 cell line. Confirmed the pattern of apoptotic cell by AO/PI double stain test and quantified apoptotic cells by Annexin V. For the cell senescence analysis, SA-β-gal staining technique was performed at 24 h after treatments, HT-29 reached maximum senescence rate at 85.74% while SW-620 had only 17.23% of senescence. And a result of cell cycle analysis, HT-29 were decreased the number of cells in S, M/G2 phase, and increased the number of cells in G0/G1 phase. Furthermore > 50 kDa peptide fraction separated from HRBE has a potent anti-cancer cells (SW-620, IC50 = 4,908 µg/ml). In conclusion, the hydrolyzed riceberry rice bran extract can inhibit colon cancer cell lines with less effect on normal cell. The extracts could induce apoptosis process in metastatic cancer cell and induce senescence process in non-metastatic cancer cell. This observed information will be useful and applicable for medical research and colon cancer treatment in the future.

Significant Gene Biomarker Tyrosine Kinase Non-receptor 2 Mediated Cell Proliferation and Invasion in Colon Cancer

Objective: This study aimed to investigate the expression and biological functions of TNK2 and miR-125a-3p in colon cancer.Materials and methods: The expression of TNK2 and miR-125a-3p in colon cancer tissues was analyzed using data deposited on public databases including UALCAN and ONCOMINE. We verified their expression in colon cancer cell lines by RT-qPCR and western blotting. By regulating the expression of TNK2 and miR-125a-3p in colon cancer cells, their functions and potential mechanisms were explored.Results:TNK2 was overexpressed in colon cancer cell lines, and it was found to directly bind to miR-125a-3p, which was downregulated in these cell lines. Their expression affected the proliferation and invasion of colon cancer cells. Additionally, colon cancer patients with lower TNK2 expression had better prognoses than those with higher TNK2 expression.Conclusion: Our results indicated that TNK2 and miR-125a-3p play critical roles in colon cancer, and could also serve as biomarkers for the diagnosis and prognosis of this malignant disease.