Human Colonic Epithelial Cells Research Articles

MTOR inhibition suppresses Myc-driven polyposis by inducing immunogenic cell death

Myc is a key driver of colorectal cancer initiation and progression, but remains a difficult drug target. In this study, we show that mTOR inhibition potently suppresses intestinal polyp formation, regresses established polyps, and prolongs lifespan of APCMin/+ mice. Everolimus in diet strongly reduces p-4EBP1, p-S6, and Myc levels, and induces apoptosis of cells with activated β-catenin (p-S552) in the polyps on day 3. The cell death is accompanied by ER stress, activation of the extrinsic apoptotic pathway, innate immune cell recruitment, and followed by T-cell infiltration on day 14 persisting for months thereafter. These effects are absent in normal intestinal crypts with physiologic levels of Myc and a high rate of proliferation. Using normal human colonic epithelial cells, EIF4E S209A knockin and BID knockout mice, we found that local inflammation and antitumor efficacy of Everolimus requires Myc-dependent induction of ER stress and apoptosis. These findings demonstrate mTOR and deregulated Myc as a selective vulnerability of mutant APC-driven intestinal tumorigenesis, whose inhibition disrupts metabolic and immune adaptation and reactivates immune surveillance necessary for long-term tumor control.

Discovery of novel benzamide derivatives bearing benzamidophenyl and phenylacetamidophenyl scaffolds as potential antitumor agents via targeting PARP-1.

Poly(ADP-ribose) polymerase-1 (PARP-1) plays a crucial role in DNA damage repair and has been identified as a promising therapeutic target in cancer therapy. As a continuation of our efforts on the development of novel PARP-1 inhibitors with potent anticancer activity, a series of benzamide derivatives containing the benzamidophenyl and phenylacetamidophenyl scaffolds were designed and synthesized based on the structure optimization of our previously reported compound IX. All target compounds were screened for their in vitro antiproliferative activities against human colorectal cancer cells (HCT116, DLD-1 and SW480) and human normal colonic epithelial cells (NCM460). Among them, compound 13f exhibited the most potent anticancer activity against HCT116cells and DLD-1cells with IC50=0.30μM and 2.83μM, respectively. Moreover, 13f displayed significant selectivity in inhibiting HCT116 cancer cells over the normal NCM460cells. Furthermore, 13f exhibited excellent PARP-1 inhibitory effect with IC50=0.25nM. Besides, 13f was found to effectively inhibit colony formation and migration of HCT116cells. Studies on the mechanisms revealed that 13f could arrest cell cycle at G2/M phase, accumulate DNA double-strand breaks, reduce mitochondrial membrane potential and ultimately induce apoptosis in HCT116cells. In addition, molecular docking study indicated that 13f could combine firmly with the catalytic pocket of PARP-1 through multiple hydrogen bond interactions. Collectively, these findings demonstrated that 13f could serve as a promising anticancer candidate and deserves further investigation.

IFN-γ is Necessary but not Sufficient to mediate Activated CD4+ T cell-induced epithelial cell damage and death

Abstract Bidirectional communication between the intestinal epithelial barrier and the immune cells in the underlying lamina propria is critical to human health. This study investigates the impact of human T cell subtypes on intestinal epithelial cell phenotype and survival. Blood-derived CD4+ or CD8+ T cells were isolated from healthy human participants using negative magnetic separation and co-cultured with healthy human colonic epithelial stem cell spheroids suspended in Matrigel. A subset of T cells was activated by anti-CD3/CD28 cross-linking for 24 h prior to co-culture. Only activated CD4+ T cells (ACD4) caused significant epithelial cell damage and death as measured by histological scoring of brightfield microscopic images and zombie violet+ epithelial cells detected by flow cytometry. The mechanism of cell damage and death was determined to be via soluble mediators, in that conditioned media from ACD4 but not activated CD8+ T cells (ACD8) significantly induced spheroid cell damage and death. A Luminex cytokine multiplex assay revealed that high levels of IFN-γ, IL-17, and TNF-α were produced by ACD4 as compared with ACD8. Neutralizing IFN-γ antibody completely protected spheroids from death induced by ACD4. In contrast, the addition of only IFN-γ protein did not induce spheroid cell death. This finding conflicts with currently published literature that indicates IFN-γ protein is able to induce spheroid cell death for both humans and mice. In addition, ACD8 also secrete IFN-γ, although to a lesser extent than ACD4, yet do not initiate spheroid cell death. These results indicate that there is a unique mixture of immune mediator released by ACD4s that mediate spheroid damage and death. DP1 DA-037997 T32 GM007250

Promotion of hair growth by Lactobacillus and fermented traditional Korean berry in C57BL/6 mice

Hair loss is the state of losing hair from the scalp or entire body when new hair does not replace the hair that has fallen out. Currently, remedies such as oral finasteride and topical minoxidil are commonly used. However, these drugs may have temporary effects and cause side effects such as depression, irregular heartbeat and weight gain when used long term. Therefore, an alternative hair growth promoting agent is needed. The hair follicle undergoes anagen, catagen, and telogen phase which affect hair growth, and this hair-growth cycle is regulated by growth factors, cytokines, and hormones. Some studies reported that the gut microbiome (GM) regulates the transition between the hair growth cycle by producing hair growth-related factors and nutrients. Also, inflammation by GM affects the cascade of the inflammation in the hair follicle, which may lead to hair growth arrest. Previous studies showed strains of Lactobacillus (LAB) have anti-oxidative and anti-inflammatory effects in an HT-29 human colon epithelial cell model and an HaCaT human keratinocyte cell model. Also, traditional Korean berry has anti-inflammatory effects in an HT-29 model. Thus, LAB and fermented traditional Korean berry by LAB (FB) may promote hair growth by regulating gut microbiome. This study investigated the effect of LAB and FB on hair growth in vivo. Six-week-old male C57BL/6 mice were used for evaluating the hair growth-promoting effects of the LAB and FB. The dorsal hair of mice was depilated, and they were orally treated with LAB at 1010 CFU/kg and FB at 250mg/kg daily for 22 days. As a result, the hair re-growth rate was significantly increased in LAB and FB groups compared with the control group. In the histological analysis of skin, FB increased the number of hair follicle and subcutaneous fat layer thickness. The mRNA expression levels of hair growth-related growth factors such as VEGF and IGF-1 are significantly increased in colon and skin. Moreover, Treatment of LAB and FB changed the composition of the microbiome and short chain fatty acids in mice. Altogether, LAB and FB supplementation may promote hair growth by regulating growth factors and altering gut microbiota compositions. In conclusion, strain of LAB and FB are potential novel approaches for hair growth promotion. This work was supported by Korea Testing Laboratory This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The role and molecular mechanism of metabolic reprogramming of colorectal cancer by UBR5 through PYK2 regulation of OXPHOS expression study.

Colorectal carcinoma (CRC) is the third most malignant tumor in the world, but the key mechanisms of CRC progression have not been confirmed.UBR5 and PYK2 expression levels were detected by RT-qPCR. The levels of UBR5, PYK2, and mitochondrial oxidative phosphorylation(OXPHOS) complexes were detected by western blot analysis. Flow cytometry was used to detect ROS activity. The CCK-8 assay was used to assess cell proliferation and viability. The interaction between UBR5 and PYK2 was detected by immunoprecipitation. A clone formation assay was used to determine the cell clone formation rate. The ATP level and lactate production of each group of cells were detected by the kit. EdU staining was performed for cell proliferation.Transwell assay was performed for cell migration ability. For the CRC nude mouse model, we also observed and recorded the volume and mass of tumor-forming tumors.The expression of UBR5 and PYK2 was elevated in both CRC and human colonic mucosal epithelial cell lines, and knockdown of UBR5 had inhibitory effects on cancer cell proliferation and cloning and other behaviors in the CRC process by knockdown of UBR5 to downregulate the expression of PYK2, thus inhibiting the OXPHOS process in CRC; rotenone (OXPHOS inhibitor) treatment enhanced all these inhibitory effects.Knockdown of UBR5 can reduce the expression level of PYK2, thus downregulating the OXPHOS process in CRC cell lines and inhibiting the CRC metabolic reprogramming process.

Quassinoid analogs exert potent antitumor activity via reversible protein biosynthesis inhibition in human colorectal cancer

Cellular protein synthesis is accelerated in human colorectal cancer (CRC), and high expression of protein synthesis regulators in CRC patients is associated with poor prognosis. Thus, inhibition of protein synthesis may be an effective therapeutic strategy for CRC. We previously demonstrated that the quassinoid bruceantinol (BOL) had antitumor activity against CRC. Herein, potent tumor growth suppression (>80%) and STAT3 inhibition was observed in two different mouse models following BOL administration. Loss of body and spleen weight was observed but was eliminated upon nanoparticle encapsulation while maintaining strong antitumor activity. STAT3 siRNA knockdown exhibited modest suppression of cell proliferation. Surprisingly, STAT3 inhibition using a PROTAC degrader (SD-36) had little effect on cancer cell proliferation suggesting the possibility of additional mechanism(s) of action for quassinoids. BOL-resistant (BR) cell lines, HCT116BR and HCA7BR, were equally sensitive to standard CRC therapeutic agents and known STAT3 inhibitors but resistant to homoharringtonine (HHT), a known protein synthesis inhibitor. The ability of quassinoids to inhibit protein synthesis was dependent on the structure of the C15 sidechain. Of note, BOL did not inhibit protein synthesis in normal human colon epithelial cells whereas HHT and napabucasin remained effective in these normal cells. Novel quassinoids were designed, synthesized, and evaluated in pre-clinical CRC models. Treatment with the most potent analog, 5c, resulted in significant inhibition of cell proliferation and protein synthesis at nanomolar concentrations. These quassinoid analogs may represent a novel class of protein synthesis inhibitors for the treatment of human CRC.

Purification of phenoloxidase from Haliotis discus hannai and its anti-inflammatory activity in vitro

Haliotis discus hannai, a food with a high protein content, is widely consumed in Asian countries. It is known to have antioxidant, anticancer, and antibacterial effects. Since the biological significance of H. discus hannai hemolymph has not been widely studied, the objective of the present study was to purify phenoloxidase (PO) and investigate its immunological effects on human colonic epithelial cells. PO was purified through ammonium sulfate precipitation and one step column chromatography. The molecular weight of the protein was about 270 kDa. When PO was mixed with Gram-negative bacteria-derived lipopolysaccharide (LPS) at various ratios (10:1–1:10, w/w), the amount of residual LPS was reduced. PO at concentrations up to 200 μg/mL was not cytotoxic to HT-29 cells. The inflammatory response induced by LPS in HT-29 cells was regulated when the concentration of PO was increased. With increasing concentration of PO, production levels of pro-inflammatory cytokines, cytokines associated with hyperimmune responses such as IL4, IL-5, and INF-γ, and prostaglandin 2 (PGE2) were regulated. It was thought that simultaneous treatment with PO and LPS anti-inflammatory effects in HT-29 cells showed by regulating the ERK1/2-mediated NF-κB pathway. Results of this study suggest that H. discus hannai hemolymph is involved in the regulation of Gram-negative bacteria-related inflammatory immune responses in human colonic epithelial cells.

Chemopreventive potential of kimchi, an ethnic food from Korea, against colorectal carcinogenesis associated with red meat intake

Kimchi, the traditional fermented vegetables with seasonings from Korea, is globally renowned as healthy food with anticancer properties. Colorectal cancer is the world’s third leading cancer with an increasing incidence rate over the past years. High red meat intake is strongly associated with colorectal cancer, and the World Health Organization has identified red meat as probably carcinogenic to humans (Group 2A). This study aimed to investigate the chemopreventive potential of cabbage kimchi against the promotion of colorectal cancer by red meat in vivo using carcinogen-injected rats. Rats were fed by AIN-76 diet enriched in freeze-dried beef sirloin supplemented or not with kimchi powder for 120 days prior to killing. Kimchi supplementation at moderate (5%) and high (10%) concentration significantly suppressed the formation of precancerous lesions in the colon of red meat-fed rats. Using fecal water, the aqueous extract of feces, we found out that kimchi supplementation limited iron-mediated oxidation and reduced lipid peroxidation in the feces of rats. Furthermore, kimchi supplementation reduced the toxicity of fecal water of red meat-fed rats toward human colonic epithelial cells in vitro by suppressing the formation of cellular reactive oxygen species (ROS). The chemopreventive effects of kimchi were associated with the acidification of fecal matrix and increase in fecal lactic acid bacteria (LAB). Gene expression analysis in the colon of rats demonstrated that kimchi supplementation prevented colorectal carcinogenesis by up-regulating the expression of tumor-suppressor genes and antioxidant enzymes, as well as by down-regulating the expression of proinflammatory proteins. Taken together, our findings suggested that kimchi consumption is correlated with lower promotion of colorectal cancer associated with red meat intake.

Proliferation Inhibitory Activity of Quinones from Blaps rynchopetera Defense Secretion on Colorectal Tumor Cells.

To explore the proliferation inhibitory effect of quinones from Blaps rynchopetera defense secretion on colorectal tumor cell lines. Human colorectal cancer cell HT-29, human colorectal adenocarcinoma cell Caco-2 and normal human colon epithelial cell CCD841 were chosen for the evaluation of inhibitory activity of the main quinones of B. rynchopetera defense secretion, including methyl p-benzoquinone (MBQ), ethyl p-benzoquinone (EBQ), and methyl hydroquinone (MHQ), through methyl thiazolyl tetrazolium assay. The tumor-related factors, cell cycles, related gene expressions and protein levels were detected by enzyme-linked immunosorbent assy, flow cytometry, RT-polymerase chain reaction and Western blot, respectively. MBQ, EBQ, and MHQ could significantly inhibit the proliferation of Caco-2, with half maximal inhibitory concentration (IC50) values of 7.04 ± 0.88, 10.92 ± 0.32, 9.35 ± 0.83, HT-29, with IC50 values of 14.90 ± 2.71, 20.50 ± 6.37, 13.90 ± 1.30, and CCD841, with IC50 values of 11.40 ± 0.68, 7.02 ± 0.44 and 7.83 ± 0.05 µg/mL, respectively. Tested quinones can reduce the expression of tumor-related factors tumor necrosis factor α, interleukin (IL)-10, and IL-6 in HT-29 cells, selectively promote apoptosis, and regulate the cell cycle which can reduce the proportion of cells in the G1 phase and increase the proportion of the S phase. Meanwhile, tested quinones could up-regulate mRNA and protein expression of GSK-3β and APC, while down-regulate that of β-catenin, Frizzled1, c-Myc, and CyclinD1 in the Wnt/β-catenin pathway of HT-29 cells. Quinones from B. rynchopetera defense secretion could inhibit the proliferation of colorectal tumor cells and reduce the expression of related factors, which would be functioned by regulating cell cycle, selectively promoting apoptosis, and affecting Wnt/β-catenin pathway-related mRNA and protein expressions.

Abstract 1215: Synthetic colibactins reveal the structural and biological mode-of-action of a microbial carcinogen

Abstract Certain Enterobacteriaceae strains contain a 54-kb biosynthetic gene cluster referred to as “pks” encoding the biosynthesis of a secondary metabolite, colibactin, that putatively alkylates host DNA via nucleophilic cyclopropane ring addition. While evidence suggests colibactin’s genotoxicity promotes mutagenesis and cancer, it has been impossible to directly test this hypothesis due to compound instability and sparse production. Recently synthesized stable colibactin analogs facilitate the first studies investigating the compounds structural mode of action. We tested the genotoxic capacity of two compounds: colibactin 742 and an analogous molecule with modified proposed DNA-binding residues (colibactin 746), in human cell lines and colonic organoids. To assess transcriptional pathways associated with acute colibactin response, we compared transcriptional profiles in a normal human colonic cell line (FHC) after short-term exposure to colibactin 742 or 746 and pks+ K. pneumoniae or an isogenic ΔclbP mutant. To determine the mutagenic and transformative potential of colibactin 742, we exposed the human colonic epithelial cell line HCT 116 to 742 or 746 for 32-51 days followed by a 30-day recovery period. We assessed changes in gene expression by RNAseq and mutagenesis by whole-genome sequencing followed by mutational signature fitting. 742 but not 746 caused DNA damage in cell lines and human colonoids, quantified by γH2AX phosphorylation. 742 recapitulated transcriptomic responses to pks+ K. pneumoniae infection, and significantly upregulated pathways associated with p53 signaling, senescence, and IL-1 signaling, relative to 746 treated cells. Moreover, chronic cyclical exposure significantly increased expression of BRCA1-associated DNA repair pathways and genes involved in cross-link repair or replication-associated DNA damage response, consistent with colibactin’s putative cross-linking activity. Whole-genome sequencing revealed an increase in single-base substitutions (SBS) and indels (ID) in 742 treated cells, with a higher proportion of [T>N] SBS and 1-4 bp insertion/deletions in T-rich homopolymers, respectively. We found that 742-induced mutations could be attributed to the proposed colibactin signature (SBS88), reactive oxygen species (ROS; SBS17), or mismatch-repair deficiency (MMRd; SBS44). Colibactin 742 treatment caused an increase in H2DCFDA staining in intestinal epithelial cells, and an increase in transcriptomic pathways associated with peroxisome biogenesis or oxidative phosphorylation, supporting the theory that colibactin 742 treatment caused ROS-induced mutations. ID signatures primarily recapitulated pks-associated mutations. These findings provide the first direct evidence supporting colibactin’s proposed mode-of-action. Furthermore, our study suggests colibactin may indirectly cause mutations attributed to ROS or MMRd. Citation Format: Michael W. Dougherty, Rafael Valdés-Mas, Kevin M. Wernke, Raad Z. Gharaibeh, Alberto Riva, Ye Yang, Marcus Muehlbauer, Eran Elinav, Jens Puschhof, Seth B. Herzon, Christian Jobin. Synthetic colibactins reveal the structural and biological mode-of-action of a microbial carcinogen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1215.

Paris polyphylla Sm. Induces Reactive Oxygen Species and Caspase 3-Mediated Apoptosis in Colorectal Cancer Cells In Vitro and Potentiates the Therapeutic Significance of Fluorouracil and Cisplatin.

Paris polyphylla Sm. (Melanthiaceae) is an essential, vulnerable herb with a wide range of traditional applications ranging from fever to cancer in various communities. The use of P. polyphylla in India is limited to traditional healers. Here, we demonstrated that P. polyphylla extract (PPE) has good phenol, flavonoid, saponin, and steroidal saponin content and anti-oxidant activity with IC50 35.12 ± 6.1 μg/mL in DPPH and 19.69 ± 6.7 μg/mL in ABTS. Furthermore, PPE induces cytotoxicity in HCT-116 with IC50 8.72 ± 0.71 μg/mL without significant cytotoxicity inthe normal human colon epithelial cell line, CCD 841 CoN. PPE inhibits the metastatic property and induces apoptosis in HCT-116, as measured by Annexin V/PI, by increasing the production of reactive oxygen species (ROS) and caspase 3 activation. PPE acts synergistically with 5FU and cisplatin in HCT-116 and potentiates their therapeutic significance. Steroidal saponins with anticancer activities were detected in PPE by HR-LCMS. The present study demonstrated that PPE induces apoptosis by increasing ROS and activating caspase 3, which was attributed to steroidal saponins. PPE can be used as a potential natural remedy for colon cancer.

Characterization and utility of two monoclonal antibodies to cholera toxin B subunit

Cholera toxin B subunit (CTB) is a potent immunomodulator exploitable in mucosal vaccine and immunotherapeutic development. To aid in the characterization of pleiotropic biological functions of CTB and its variants, we generated a panel of anti-CTB monoclonal antibodies (mAbs). By ELISA and surface plasmon resonance, two mAbs, 7A12B3 and 9F9C7, were analyzed for their binding affinities to cholera holotoxin (CTX), CTB, and EPICERTIN: a recombinant CTB variant possessing mucosal healing activity. Both 7A12B3 and 9F9C7 bound efficiently to CTX, CTB, and EPICERTIN with equilibrium dissociation constants at low to sub-nanomolar concentrations but bound weakly, if at all, to Escherichia coli heat-labile enterotoxin B subunit. In a cyclic adenosine monophosphate assay using Caco2 human colon epithelial cells, the 7A12B3 mAb was found to be a potent inhibitor of CTX, whereas 9F9C7 had relatively weak inhibitory activity. Meanwhile, the 9F9C7 mAb effectively detected CTB and EPICERTIN bound to the surface of Caco2 cells and mouse spleen leukocytes by flow cytometry. Using 9F9C7 in immunohistochemistry, we confirmed the preferential localization of EPICERTIN in colon crypts following oral administration of the protein in mice. Collectively, these mAbs provide valuable tools to investigate the biological functions and preclinical development of CTB variants.

Apigenin reduces the suppressive effect of exosomes derived from irritable bowel syndrome patients on the autophagy of human colon epithelial cells by promoting ATG14

BackgroundInflammatory bowel disease (IBS) is a chronic disorder of the gastrointestinal tract. Exosomes have been involved in various pathological processes including IBS. Apigenin has been reported to suppress inflammatory bowel disease (IBS). However, the regulatory roles of exosomes derived from IBS patients (IBS-exos) on human colon epithelial cells are still unclear.MethodsExosomes were collected from IBS patients (IBS-exos) and co-cultured with CACO-2 cells. Apigenin was used to treat IBS-exos-treated CACO-2 cells. By exploring the public data bank, we figured out the regulators control the autophagy of CACO-2 cells.ResultsAdministration of apigenin dose-dependently abolished the inhibitory effect of IBS-exo on the autophagy of CACO-2 cells. A mechanistic study showed that miR-148b-3p bound to 3′UTR to suppress ATG14 and decrease autophagy. Moreover, results suggested that ATG14 overexpression promoted the autophagy of CACO-2 cells in the presence of miR-148b-3p mimic.ConclusionThe current study showed that apigenin dose-dependently abolished the inhibitory effect of IBS-exo on CACO-2 cell autophagy by regulating miR-148b-3p/ATG14 signaling.

Phytochemical Profiling of Extracts from Rare Potentilla Species and Evaluation of Their Anticancer Potential.

Despite the common use of Potentilla L. species (Rosaceae) as herbal medicines, a number of species still remain unexplored. Thus, the present study is a continuation of a study evaluating the phytochemical and biological profiles of aqueous acetone extracts from selected Potentilla species. Altogether, 10 aqueous acetone extracts were obtained from the aerial parts of P. aurea (PAU7), P. erecta (PER7), P. hyparctica (PHY7), P. megalantha (PME7), P. nepalensis (PNE7), P. pensylvanica (PPE7), P. pulcherrima (PPU7), P. rigoi (PRI7), and P. thuringiaca (PTH7), leaves of P. fruticosa (PFR7), as well as from the underground parts of P. alba (PAL7r) and P. erecta (PER7r). The phytochemical evaluation consisted of selected colourimetric methods, including total phenolic (TPC), tannin (TTC), proanthocyanidin (TPrC), phenolic acid (TPAC), and flavonoid (TFC) contents, as well as determination of the qualitative secondary metabolite composition by the employment of LC-HRMS (liquid chromatography-high-resolution mass spectrometry) analysis. The biological assessment included an evaluation of the cytotoxicity and antiproliferative properties of the extracts against human colon epithelial cell line CCD841 CoN and human colon adenocarcinoma cell line LS180. The highest TPC, TTC, and TPAC were found in PER7r (326.28 and 269.79 mg gallic acid equivalents (GAE)/g extract and 263.54 mg caffeic acid equivalents (CAE)/g extract, respectively). The highest TPrC was found in PAL7r (72.63 mg catechin equivalents (CE)/g extract), and the highest TFC was found in PHY7 (113.29 mg rutin equivalents (RE)/g extract). The LC-HRMS analysis showed the presence of a total of 198 compounds, including agrimoniin, pedunculagin, astragalin, ellagic acid, and tiliroside. An examination of the anticancer properties revealed the highest decrease in colon cancer cell viability in response to PAL7r (IC50 = 82 µg/mL), while the strongest antiproliferative effect was observed in LS180 treated with PFR7 (IC50 = 50 µg/mL) and PAL7r (IC50 = 52 µg/mL). An LDH (lactate dehydrogenase) assay revealed that most of the extracts were not cytotoxic against colon epithelial cells. At the same time, the tested extracts for the whole range of concentrations damaged the membranes of colon cancer cells. The highest cytotoxicity was observed for PAL7r, which in concentrations from 25 to 250 µg/mL increased LDH levels by 145.7% and 479.0%, respectively. The previously and currently obtained results indicated that some aqueous acetone extracts from Potentilla species have anticancer potential and thus encourage further studies in order to develop a new efficient and safe therapeutic strategy for people who have been threatened by or suffered from colon cancer.

Intestinal Epithelial Cells Adapt to Chronic Inflammation through Partial Genetic Reprogramming.

Reactive oxygen species (ROS) are considered to be the main drivers of inflammatory bowel disease. We investigated whether this permanent insult compels intestinal stem cells to develop strategies to dampen the deleterious effects of ROS. As an adverse effect, this adaptation process may increase their tolerance to oncogenic insults and facilitate their neoplastic transformation. We submitted immortalized human colonic epithelial cells to either a mimic of chronic inflammation or to a chemical peroxide, analyzed how they adapted to stress, and addressed the biological relevance of these observations in databases. We demonstrated that cells adapt to chronic-inflammation-associated oxidative stress in vitro through a partial genetic reprogramming. Through a gene set enrichment analysis, we showed that this program is recurrently active in the intestinal mucosae of Crohn's and ulcerative colitis disease patients and evolves alongside disease progression. Based on a previously reported characterization of intestinal stem and precursor cells using tracing experiments, we lastly confirmed the activation of the program in intestinal precursor cells during murine colorectal cancer development. This adaptive process is thus likely to play a role in the progression of Crohn's and ulcerative disease, and potentially in the initiation of colorectal cancer.

P019 Host-Enterobacteriaceae interactions and mitochondrial dysfunction in Crohn’s colitis

Abstract Background Crohn’s disease (CD) is characterized by chronic gastrointestinal inflammation and unfavourable changes in the composition of intestinal microbiota (‘dysbiosis’). Members of Enterobacteriaceae such as K. pneumoniae and E. coli are often over-represented in colonic mucosa of CD patients. These bacteria contribute to vicious circle of dysbiosis and inflammation by expressing multiple virulence factors. Further, mitochondrial dysfunction in Paneth cells has recently been reported in ileal CD. In agreement, high-fat diet and repeated use of antibiotics increases susceptibility to colitis by damaging mitochondria. However, it remains unknown whether impaired mitochondrial bioenergetics could enhance mucosal colonization by Enterobacteriaceae and thus, contribute to the pathophysiology of colonic CD. Furthermore, the molecular mechanism leading to mitochondrial damage in colonic CD are incompletely understood. Methods Right colonic biopsies were collected from 74 adult CD patients and 28 healthy participants at St. Paul’s Hospital (Vancouver, BC, Canada). Microbes in colonic biopsies were characterized by 16S rDNA sequencing using Illumina MiSeq platform, followed by analysis of differentially abundant families via ANCOM-BC methodology. Further, high-throughput data-independent acquisition (DIA) proteomics was performed in colonic biopsies (Bruker Daltonics TimsTof Pro2 LC-MS/MS), and later analyzed via DIA-neural network software. Spearman's rank correlation approach was used for host proteomics- Enterobacteriaceae correlational analysis. Microbiome and proteomic data were corrected for false discovery rate (FDR; < 0.5 for statistical significance). For validation studies, human colonic TC7 epithelial cells and patient-derived organoid cultures were infected with CD-patient derived E. coli ± C75, a chemical inhibitor of fatty acid synthase (FASN; de novo fatty acid synthesis enzyme). Results We found an increased abundance of Enterobacteriaceae members in colonic mucosa of CD patients compared to healthy controls (Fig A). Next, our proteomics data revealed a significant downregulation in a group of proteins which were functionally related to mitochondrial health and activity, in CD patients compared to controls (Fig B). Further, Spearman’s analysis showed a positive correlation between Enterobacteriaceae and host FASN enzyme (Fig C), a protein directly related to mitochondrial metabolism. Interestingly, we found that blocking FASN with chemical inhibitor (C75) significantly enhanced colonization by E. coli in TC7 cells (Fig D). Conclusion Mechanistically, our study reports that aberrant activity of host FASN enzyme may cause mitochondrial dysfunction and thus, facilitates intestinal colonization by Enterobacteriaceae in CD patients.

A NOVEL DRUG THERAPY INDUCES MITOCHONDRIAL BIOGENESIS AND ATTENUATES COLITIS

Abstract Mitochondrial dysfunction is a characteristic feature of intestinal epithelium cells (IEC) in IBD patients, associated with oxidative stress and impaired ATP production. We posit that disrupted mitochondrial bioenergetics impairs ulcer healing in IBD. In the current study, we tested a novel gold-based compound (AuPhos) that improves mitochondrial function by increasing mitochondrial biogenesis and antioxidant enzymes in IEC and human colonic biopsies. METHODS: Normal human colonic epithelial cells (NCM460) were used to evaluate the effect of AuPhos on mitochondrial proteins and anti-oxidant proteins by western blotting in the presence or absence of low-dose TNF (1ng/ml). To model mitochondrial deficiency, TFAM (mitochondrial transcriptional factor A) was silenced (shTFAM) in NCM460 cells. These cells were treated with low-dose TNF to mimic inflammatory conditions in the human intestine. The mitochondrial mass was assessed by flow cytometry using mito tracker green. The transcriptional activity of genes associated with mitochondrial biogenesis, mitochondrial complexes, and anti-oxidants was evaluated using RT-qPCR in AuPhos or vehicle-treated (overnight) cells with or without TNF. Human colonic biopsy samples were collected in Allprotect, treated ex-vivo with vehicle or AuPhos (3h, 4oC), and processed for RNA isolation followed by RT-qPCR. RESULTS: Biochemical and mass spectrometry analyses revealed that AuPhos localizes to the mitochondria. We show here that AuPhos increases mitochondrial mass and mRNA for mitochondrial complexes [complex IV (Cox6A1) and complex V (ATP5A1)] in NCM460 cells in TNF or vehicle-treated cells. Similarly, AuPhos increased protein levels of upstream regulators of mitochondrial biogenesis such as TFAM, PGC-1α, and Nrf2 compared to nontreated cells. Further protein analysis of AuPhos-treated cells showed that AuPhos increases downstream antioxidant markers such as manganese superoxide dismutase (MnSOD), peroxidane 3 (Prd3), glutathione peroxidase 4 (GPX4), thioredoxin reductase2 (Trx2), and catalase expression when compared to the TNF or control samples. Interestingly, expression of genes associated with mitochondria-specific (MnSOD) and cytoplasmic superoxide dismutase (CuZnSOD) were also significantly increased in AuPhos-treated shTFAM NCM cells compared to vehicle or TNF-treated cells. AuPhos substantially upregulates the expression of mtCO1, mtCO2, Cox6A1, ATP5A1, NRF1, MnSOD, GPX, PRDX-2,-4, and -6, in ex vivo treated human colonic biopsies compared to vehicle or TNF-treated samples. CONCLUSIONS: The current data suggest that the AuPhos compound improves mitochondrial function by promoting the expression of mitochondrial-associated proteins and antioxidant enzymes (Figure 1). We believe AuPhos can be used as a potential therapeutic in treating ulcerative colitis and Crohn’s disease by restoring mitochondrial biogenesis.

Association of GILZ with MUC2, TLR2, and TLR4 in Inflammatory Bowel Disease.

Ulcerative colitis (UC) and Crohn's Disease (CD) are chronic relapsing inflammatory diseases that are caused by genetic, environmental, and immune factors. Treatment strategies are currently based on symptomatic control by immunosuppression. The glucocorticoid-induced leucine zipper (GILZ), a mediator of several effects of glucocorticoids, was recently found to be secreted by goblet cells and play a role in inflammatory bowel disease (IBD). This study investigates which genes GILZ is associated with in its role in intestinal barrier functions. We examined datasets from the Gene Expression Omnibus (GEO) and ArrayExpress profiles of the gut of healthy subjects (HSs), as well as UC and CD patients. The human colonic epithelial HT29 cell line was used for in vitro validation experiments. GILZ was significantly correlated with MUC2, TLR2, and TLR4. In particular, an inverse correlation was found between the GILZ and MUC2 in HS and patients with IBD, mostly in those with an active disease. Further, direct pairwise correlations for GILZ/TLR2 and GILZ/TLR4 were found in HSs and UC patients, but not in CD patients. Overall, our results reveal the crosstalk at the transcription level between the GILZ, MUC2, and TLRs in the mucosal barrier through common pathways, and they open up new perspectives in terms of mucosal healing in IBD patients.

High-Throughput Screening for Epigenetic Compounds That Induce Human β-Defensin 1 Synthesis

Antimicrobial host defense peptides (HDPs) are critically important for innate immunity. Small-molecule compounds with the ability to induce HDP synthesis are being actively explored for antimicrobial therapy. To facilitate the discovery of the compounds that specifically activate human β-defensin 1 (DEFB1) gene transcription, we established a cell-based high-throughput screening assay that employs HT-29/DEFB1-luc, a stable reporter cell line expressing the luciferase gene driven by a 3-Kb DEFB1 gene promoter. A screening of a library of 148 small-molecule epigenetic compounds led to the identification of 28 hits, with a minimum strictly standardized mean difference of 3.0. Fourteen compounds were further selected and confirmed to be capable of inducing DEFB1 mRNA expression in human HT-29 colonic epithelial cells. Desirably, the human cathelicidin antimicrobial peptide (CAMP) gene was also induced by these epigenetic compounds. Benzamide-containing histone deacetylase inhibitors (HDACi) were among the most potent HDP inducers identified in this study. Additionally, several major genes involved in intestinal barrier function, such as claudin-1, claudin-2, tight junction protein 1, and mucin 2, were differentially regulated by HDP inducers. These findings suggest the potential for the development of benzamide-based HDACi as host-directed antimicrobials for infectious disease control and prevention.

Genistein Inhibits Clostridioides difficile Infection via Estrogen Receptors and Lysine-Deficient Protein Kinase 1.

Clostridioides difficile infection (CDI) is a debilitating nosocomial disease. Postmenopausal women may have an increased risk of CDI, suggesting estrogen influence. Soybean products contain a representative estrogenic isoflavone, genistein. The anti-inflammatory and antiapoptotic effects of genistein were determined using primary human cells and fresh colonic tissues. The effects of oral genistein therapy among mice and hamsters were evaluated. Within 10 days of CDI, female c57BL/6J mice in a standard environment (regular diet) had a 50% survival rate, while those with estrogen depletion and in an isoflavone-free environment (soy-free diet) had a 25% survival rate. Oral genistein improved their 10-day survival rate to 100% on a regular diet and 75% in an isoflavone-free environment. Genistein reduced macrophage inflammatory protein-1α (MIP-1α) secretion in fresh human colonic tissues exposed to toxins. Genistein inhibited MIP-1α secretion in primary human peripheral blood mononuclear cells, abolished apoptosis and BCL-2-associated X (BAX) expression in human colonic epithelial cells, and activated lysine-deficient protein kinase 1 (WNK1) phosphorylation in both cell types. The anti-inflammatory and antiapoptotic effects of genistein were abolished by inhibiting estrogen receptors and WNK1. Genistein reduces CDI disease activity by inhibiting proinflammatory cytokine expression and apoptosis via the estrogen receptor/G-protein estrogen receptor/WNK1 pathways.