Normal Colonic Cells Research Articles

Raman-AFM-fluorescence-guided impact of linoleic and eicosapentaenoic acids on subcellular structure and chemical composition of normal and cancer human colon cells

The regular overconsumption of high-energy food (rich in lipids and sugars) results in elevated nutrient absorption in intestine and consequently excessive accumulation of lipids in many organs e.g.: liver, adipose tissue, muscles. In the long term this can lead to obesity and obesity-associated diseases e.g. type 2 diabetes, non-alcoholic fatty liver disease, cardiovascular disease, inflammatory bowel disease (IBD). In the presented paper based on RI data we have proved that Raman maps can be used successfully for subcellular structures visualization and analysis of fatty acids impact on morphology and chemical composition of human colon single cells – normal and cancer. Based on Raman data we have investigated the changes related to endoplasmic reticulum, mitochondria, lipid droplets and nucleus. Analysis of ratios calculated based on Raman bands typical for proteins (1256, 1656 cm−1), lipids (1304, 1444 cm−1) and nucleic acids (750 cm−1) has confirmed for endoplasmic reticulum the increased activity of this organelle in lipoproteins synthesis upon FAs supplementation; for LDs the changes of desaturation of accumulated lipids with the highest unsaturation level for CaCo-2 cells upon EPA supplementation; for mitochondria the stronger effect of FAs supplementation was observed for CaCo-2 cells confirming the increased activity of this organelle responsible for energy production necessary for tumor development; the weakest impact of FAs supplementation was observed for nucleus for both types of cells and both types of acids. Fluorescence imaging was used for the investigations of changes in LDs/ER morphology. Our measurements have shown the increased area of LDs/ER for CaCo-2 cancer cells, and the strongest effect was noticed for CaCo-2 cells upon EPA supplementation. The increased participation of lipid structures for all types of cells upon FAs supplementation has been confirmed also by AFM studies. The lowest YM values have been observed for CaCo-2 cells including samples treated with FAs.

Abstract 5344: Myeloid populations upregulated in prostate cancer are associated with colitis induced colorectal cancer

Abstract Chronic systemic inflammation caused by diseases like ulcerative colitis (UC) and Crohn’s disease (CD) increases the risk of developing colorectal cancer (CRC). Research indicates that patients with UC are more susceptible to prostate cancer (PCa) and individuals with PCa may also be at a higher risk for developing CRC. However, these relationships are not well defined. Here, we explored these relationships using preclinical mouse models of dextran sulfate sodium (DSS)-induced colitis (DSS-UC) and in young and aged healthy wild-type mice and conditional transgenic mice harboring Pten-deficient (KO) or Pten/Trp53-deficient (DKO) PCa. After a 25-week follow-up, DSS-induced UC caused more severe colitis in prostate tumor-bearing DKO compared to healthy WT mice. None of the DSS WT mice developed tumors, whereas 75% (3/4 mice) of DSS DKO mice did, including two out of four mice that developed multiple tumors. Overall, DKO mice with prostate tumors had increased levels of CD11b+/Gr1+ in peripheral blood, prostate, and colon compared with WT mice. Quantitative immunohistochemical analyses revealed that DKO mice with prostate tumors had a significantly higher 2.8-fold increase in the levels of Gr1+ cells in the colon compared to heathy WT mice. Overall, DSS-induced colitis further increased Gr1+ cells in the colon of WT and DKO mice by 2.5- and 2-fold, respectively. Gr1+ cells comprised 3.0%, 4.7%, and 10.1% of cells in normal colon from control WT mice and inactive and active regions of the colon in DSS-exposed WT mice, respectively. In DKO mice, Gr1+ cells comprised 7.0%, 19.2%, and 23.6% of cells in normal colon from control mice and inactive and active regions of the colon in DSS-exposed mice, respectively. CD68+, which corresponds to M1 polarized macrophages comprised 0.9%, 1.8%, and 20.6% of cells in normal colon from control WT mice and inactive and active regions of the colon in DSS-exposed WT mice, respectively, However, in DKO mice CD68+ cells comprised 3.0%, 2.8% and 1.6% of cells in normal colon from control mice and inactive and active regions of the colon in DSS-exposed mice, respectively. Our study suggests that Gr1 cells that were abundant in PCa-bearing mice were also present in colons and could have contributed to CRC. Moreover, CD68+ macrophages in the colons of healthy mice may have decreased tumor promoting effects of DSS. This study provides preclinical evidence showing that systemic inflammation in the presence of an existing malignancy increases the risk of a second malignancy, in this case linking PCa to colitis-induced-CRC. Citation Format: Yurie Kura, Marco A. De Velasco, Kazuko Sakai, Mamoru Hashimoto, Syogo Adomi, Takafumi Minami, Kazuhiro Yoshimura, Kazutoshi Fujita, Kazuto Nishio, Hirotsugu Uemura. Myeloid populations upregulated in prostate cancer are associated with colitis induced colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5344.

Abstract 5990: Microtubule-associated serine/threonine kinase-likeinhibitor, AD1208, as a novel cancer therapeutics

Abstract Microtubule-associated serine/threonine kinase-like is an essential regulator of mitosis and emerged as a novel oncogenic kinase. It is upregulated in several cancer types, correlating with chromosome instability and poor patient survival. Here, we are reporting a MASTL inhibitor, AD1208, as a new cancer therapeutics.AD1208 inhibits kinase activity of MASTL with IC50 value of 7nM in competitive manner against ATP and induces apoptosis of colorectal cancer cells with IC50 of 150nM. Selectivity index is higher than 100 folds when the cytotoxicity was measured in normal colon cells. Synthetic lethality with BRCA was observed in knock out studies. Cell panel analysis, consist of 300 cell lines, indicated that the AD1208 is effective in cancers originated from stomach, colon, leukemia, lymphoma, and soft tissue. In vivo efficacy was confirmed in mouse xenograft model in dose dependent manner and synergistic effect with PARP inhibitor was observed. Kinase Assay Panel screening revealed that AD1208 is highly selective against 468 kinases. Inhibition of MASTL resulted in decreasing phosphorylated ENSA, inactivating CDK1 and arresting cell cycle progression at G2/M phase. Clinical adverse drug reactions of AD1208 were predicted by in vitro Safety Functional Panel screening to exhibit no meaningful safety-related issues. Currently, AD1208 is being conducted in IND enabling GLP-Tox studies, including genotoxicity, mutagenicity, safety pharmacology as well as in vivo safety studies. Taken together, AD1208 is identified as a novel and selective MASTL inhibitor exhibiting significant therapeutic potential to be a development candidate. Citation Format: SOONGYU CHOI, JS Ahn, KS Baek, YL Choi, MY Im, JH Kim, JW Kim, JH Khoo, SH Lee, WL Yao, Young-Whan Park. Microtubule-associated serine/threonine kinase-likeinhibitor, AD1208, as a novel cancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5990.

Abstract 5599: Role of HOX gene expression in differentiation of colon cancer stem cells

Abstract HOX genes encode a highly conserved set of transcription factors critical for stem cell (SC) function and embryonic development. Several HOX genes have also been implicated in a myriad of cancers, including colorectal cancer (CRC). However, how HOX genes play a role in SC regulation and contributes to cancer development is unclear. HOX expression is regulated by WNT, FGF, and retinoic acid (RA) signaling - pathways which also become dysregulated in cancer. Goal: To determine how regulation of HOX genes specifies differentiation of colonic SCs into specialized cell lineages and how dysregulation of HOX genes contributes to CRC development. Hypotheses: i) Each of the different cell types within human colonic epithelium has a distinct HOX gene expression signature; ii) All-trans retinoic acid (ATRA) induced differentiation of CRC cells changes HOX expression that correlates with specific HOX expression signatures in normal colon and CRC cells. Results: Immunostaining shows that specific HOX proteins (e.g., HOXA9 & HOXC9) are selectively expressed in ALDH-positive SCs and expression of these HOX genes is increased in CRC tissues compared to normal epithelium. Bioinformatics analysis predicted that these HOX genes (plus HOXA4, HOXA10, HOXC8) are regulated by RA signaling. Accordingly, we studied how ATRA influences HOX expression in CRC cells (HT29 & SW480). We found ATRA treatment: 1) Decreases proliferation and increases neuroendocrine cell (NEC) differentiation; and 2). Leads to specific changes in HOX gene expression patterns. Using NanoString profiling, we also discovered that LGR5+ SCs, ALDH+ SCs and GLP2R+ NECs have unique HOX signatures. By defining how these HOX gene expression changes in response to ATRA treatment in comparison to the HOX gene signatures of different colonic cell lineages will help us understand the role that HOX genes play in SC differentiation and how dysregulation of HOX gene expression contributes to CRC development. Citation Format: Brian T. Osmond, Caroline O. Facey, Lynn O. Opdenaker, Chi Zhang, Bruce M. Boman. Role of HOX gene expression in differentiation of colon cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5599.

Abstract 3176: Otopetrin 3 promotes cell proliferation and regulates glycolysis via interaction with MYC in colorectal cancer

Abstract Otopetrin 3 (OTOP3) stands out as a pivotal regulator of intracellular pH through its control over proton transportation into cells, affecting fundamental cellular processes. Previous studies have shown that OTOP3 is associated with the prognosis of colorectal cancer (CRC) patients. However, the mechanism underlying OTOP3 regulation remains unclear in cancer. Our investigation reveals OTOP3 as a potent oncogene in CRC, with higher OTOP3 levels correlating with diminished overall survival rates in CRC patients. Comparative analysis between cancerous and normal colon cells highlights a pronounced overexpression of OTOP3 in cancer cells. Suppression of OTOP3 expression significantly curtailed CRC cell proliferation, colony formation, migration, and invasion, concurrently inducing apoptosis and cell cycle arrest, ultimately leading to cancer cell death. Moreover, through CHX, FBS inactivation, and MG132 treatment experiments, it was confirmed that OTOP3 regulates c-Myc expression and stability through ubiquitination and has a critical impact on CRC cell proliferation and growth. Additionally, OTOP3's modulation of HK2, PKM2, and LDHA, underscores its role in impacting cancer cell behavior via glycolytic pathways. In summary, the overexpression of OTOP3 in CRC cells drives cell proliferation and growth through the regulation of c-Myc, impacting crucial cellular processes. Additionally, it promotes these processes by influencing glycolysis. These findings provide valuable insights into potential therapeutic targets for CRC treatment, possibly involving OTOP3 and its associated pathways. Citation Format: Hyeung-Jin Jang, Wona Jee, So-Mi Park, Ye-rin Park, Seok Woo Kim, Ji Hoon Jung. Otopetrin 3 promotes cell proliferation and regulates glycolysis via interaction with MYC in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3176.

High Expression Level of TRIP6 is Correlated with Poor Prognosis in Colorectal Cancer and Promotes Tumor Cell Proliferation and Migration.

Globally, colorectal cancer (CRC) is one of the leading causes of health problems. More reliable molecular biomarkers for early diagnosis in CRC patients are needed. A crucial role for thyroid hormone receptor interacting protein 6 (TRIP6) is played in tumorigenesis and tumor growth. Our study aims to determine the diagnostic and prognostic roles of TRIP6 at CRC. TRIP6 gene expression levels were analyzed in this study from public databases. The relationship between TRIP6 expression and clinicopathological characteristics was explored by logistic regression analysis. Based on Kaplan-Meier (K-M) survival curves and receiver operating characteristic curves (ROC) analysis, the prognostic and diagnostic values of TRIP6 were determined. Protein-protein interaction (PPI) networks analysis were performed using the STRING database. A Spearman's correlation analysis applied for examining the correlation between TRIP6 expression, immune cell infiltration, and immune checkpoint genes. Moreover, colony formation assay and transwell assay were used to investigate the functions of TRIP6. TRIP6 was highly expressed in CRC cancer tissues and cells. K-M survival analysis indicated that a high expression of TRIP6 was associated with poor prognosis. TRIP6 expression was obviously associated with immune cell infiltration and immune checkpoint gene expression. For validation, the results of collected clinical CRC samples show that TRIP6 levels in CRC tumor tissue were higher than those of paired adjacent colorectal tissues. Additionally, in vitro experiments suggested that TRIP6 knockdown suppressed proliferation and migration in CRC cell line RKO. TRIP6 overexpression promoted the proliferation and migration of normal colon cell line NCM460. High TRIP6 expression is associated with poor prognosis in colorectal cancer and promotes tumor cell proliferation and migration which may be a potential diagnostic and prognostic biomarker and a promising therapeutic target for CRC, providing new insights into its role in CRC.

Proximate Composition, Phytochemical Analysis and Toxicity Assessment of Extracts of Caulerpa lentillifera using Autoclave- and Microwave-Assisted Extractions

Caulerpa lentilliferais a marine alga widely cultured and consumed in Asia and Oceania. Emerging green techniques are currently popular for phytochemical extraction. Therefore, this study aimed to investigate the proximate compositions, phytochemicals, and antioxidant properties of the C. lentilliferaextracts using autoclave-assisted and microwave-assisted extraction methods (A-CLE and M-CLE, respectively). The toxicity of extracts was tested against human normal colon cells and freshwater crustacean Daphnia magna. We found that crude lipid, ash and moisture contents in A-CLE were lower than those in M-CLE, while crude protein and crude carbohydrate were higher in A-CLE. Total flavonoid content in A-CLE (1.474 ± 0.046 mg QE g-1 extract) was higher than that in M-CLE (1.179 ± 0.054 mg QE g-1 extract). There was no difference in total phenolic contents, triterpenoid contents and antioxidant activities between the extracts. The IC50 of A-CLE (12,260 ± 197 µg mL-1) was higher than that of M-CLE (10,950 ± 169 µg mL-1). In the acute toxicity test, the LC50 of A-CLE against D. magna (7.50 ± 0.28 g L-1) was lower than LC50 of M-CLE (8.76 ± 0.26 g L-1). This study suggest that autoclave-assisted and microwave-assisted extractions are effective green methods of extracting C. lentillifera. This study will be useful for further studies of C. lentillifera extracts to improve human and animal health.

The lncRNA TRG-AS1 promotes the growth of colorectal cancer cells through the regulation of P2RY10/GNA13

Background The lncRNA TRG-AS1 and its co-expressed gene P2RY10 are important for colorectal cancer (CRC) occurrence and development. The purpose of our research was to explore the roles of TRG-AS1 and P2RY10 in CRC progression. Methods The abundance of TRG-AS1 and P2RY10 in CRC cell lines (HT-29 and LoVo) and normal colon cells FHC was determined and difference between CRC cells and normal cells was compared. LoVo cells were transfected with si-TRG-AS1 and si-P2RY10 constructs. Subsequently, the viability, colony formation, and migration of the transfected cells were analyzed using cell counting kit-8, clonogenicity, and scratch-wound/Transwell® assays, respectively. Cells overexpressing GNA13 were used to further explore the relationship between TRG-AS1 and P2RY10 along with their downstream functions. Finally, nude mice were injected with different transfected cell types to observe tumor formation in vivo. Results TRG-AS1 and P2RY10 were significantly upregulated in HT-29 and LoVo compared to FHC cells. TRG-AS1 knockdown and P2RY10 silencing suppressed the viability, colony formation, and migration of LoVo cells. TRG-AS1 knockdown downregulated the expression of P2RY10, GNA12, and GNA13, while P2RY10 silencing downregulated the expression of TRG-AS1, GNA12, and GNA13. Additionally, GNA13 overexpression reversed the cell growth and gene expression changes in LoVo cells induced by TRG-AS1 knockdown or P2RY10 silencing. In vivo experiments revealed that CRC tumor growth was suppressed by TRG-AS1 knockdown and P2RY10 silencing. Conclusions TRG-AS1 knockdown repressed the growth of HT-29 and LoVo by regulating P2RY10 and GNA13 expression.

Osthole impairs mitochondrial metabolism and the autophagic flux in colorectal cancer

BackgroundOsthole is active constituent of Cnidium monnieri (L.) Cuss. with various physiological functions including anti-inflammation and anti-lipedemic effects. However, the regulatory activity of osthole in colorectal cancer development, focusing on mitochondrial metabolism, is not well known. Hypothesis/purposeWe hypothesized that osthole may suppress progression of colorectal cancer and aimed to determine the underlying mitochondrial metabolism and the autophagic flux. Study designIn this study, we elucidated the mechanism of action of osthole in colorectal cancer using an in vivo azoxymethane/dextran sodium sulfate (AOM/DSS) mouse model and an in vitro cell culture system. MethodsAOM/DSS mouse model was established and analyzed the effects of osthole on survival rate, diseases activity index, number of tumor and histopathology. Then, cell based assays including viability, cell cycle, reactive oxygen species (ROS), apoptosis, calcium efflux, and mitochondrial function were analyzed. Moreover, osthole-mediated signaling was demonstrated by western blot analyses. ResultsOsthole effectively suppressed the growth of colorectal tumors and alleviated AOM/DSS-induced intestinal injury. Osthole restored the function of goblet cells and impaired the expression of Claudin1 and Axin1 impaired by AOM/DSS. In addition, osthole specifically showed cytotoxicity in colorectal carcinoma cells, but not in normal colon cells. Osthole decreased the ASC/caspase-1/IL-1β inflammasome pathway and induced mitochondrial dysfunction in redox homeostasis, calcium homeostasis. Furthermore, osthole inhibited both oxidative phosphorylation (OXPHOS) and glycolysis, leading to the suppression of ATP production. Moreover, via combination treatment with chloroquine (CQ), we demonstrated that osthole impaired autophagic flux, leading to apoptosis of HCT116 and HT29 cells. Finally, we elucidated that the functional role of tiRNAHisGTG regulated by osthole directly affects the cellular fate of colon cancer cells. ConclusionThese results suggest that osthole has the potential to manage progression of colorectal cancer by regulating autophagy- and mitochondria-mediated signal transduction.

Design and synthesis of quinazolin-4-one derivatives as potential anticancer agents and investigation of their interaction with RecQ helicases

The upregulation of RecQ helicases has been associated with cancer cell survival and resistance to chemotherapy, making them appealing targets for therapeutic intervention. In this study, twenty-nine novel quinazolinone derivatives were designed and synthesized. The anti-proliferative activity of all compounds was evaluated against 60 cancer cell lines at the National Cancer Institute Developmental Therapeutic Program, with six compounds (11f, 11g, 11k, 11n, 11p, and 11q) being promoted to a five-dose screen. Compound 11g demonstrated high cytotoxic activity against all examined cell lines. The compounds were further assayed for Bloom syndrome (BLM) helicase inhibition, where 11g, 11q, and 11u showed moderate activity. These compounds were counter-screened against WRN and RECQ1 helicases, where 11g moderately inhibited both enzymes. An ATP competition assay confirmed that the compounds bound to the ATP site of RecQ helicases, and molecular docking simulations were used to study the binding mode within the active site of BLM, WRN, and RECQ1 helicases. Compound 11g induced apoptosis in both HCT-116 and MDA-MB-231 cell lines, but also caused an G2/M phase cell cycle arrest in HCT-116 cells. This data revealed the potential of 11g as a modulator of cell cycle dynamics and supports its interaction with RecQ helicases. In addition, compound 11g displayed non-significant toxicity against FCH normal colon cells at doses up to 100 µM, which confirming its high safety margin and selectivity on cancer cells. Overall, these findings suggest compound 11g as a potential pan RecQ helicase inhibitor with high anticancer potency and a favorable safety margin and selectivity.

In-vitro Studies of Extracts of Plumbago Zeylanica in Cancer Cell Lines.

Despite increased use of early detection methods and more aggressive treatment strategies, the worldwide incidence of colorectal cancer is still on the rise. Consequently, it remains urgent to identify novel agents with enhanced efficacy in prevention and/or therapeutic protocols. Our studies focused on the use of Plumbagin, a natural phytochemical that showed promising results against other tumor types, to determine its effectiveness in blocking the proliferation and survival of colon cancer cells in experimental protocols mimicking the environment in primary tumors (attached culture conditions) and in circulating tumor cells (unattached conditions). Under both experimental settings, exposure of HCT116 cells to Plumbagin concentrations in the low micromolar range resulted in cell cycle arrest at the G1 phase, apoptosis via the mitochondrial cell death pathway, and increased production of reactive oxygen species. The cell cycle effects were more noticeable in attached cells, whereas the induction of cell death was more evident in unattached cells. These effects were consistent with the nature and the magnitude of the alterations induced by Plumbagin on the expression levels of a set of proteins known to play key roles in the regulation of cell cycle dynamics, apoptosis mechanisms and cell proliferation. In light of its previously reported lack of toxicity on normal colon cells and the striking anti-survival effect on colon cancer cells observed in our study, Plumbagin should be considered a promising drug for the treatment of colon cancer.

Hypomethylation of Sonic hedgehog in colorectal cancer

Background: Past research has demonstrated that a changed Sonic Hedgehog (SHH) pattern contributes to the development of colorectal cancer. This study’s objective was to determine whether the SHH gene’s promoter hypomethylation might be used to predict the likelihood of developing colorectal cancer.
 Methods: For the current investigation, 50 newly diagnosed and untreated colorectal cancer patients’ tumor samples and surrounding non-tumor tissues were gathered. SHH methylation was determined by methylation-specific PCR (MSP) and results correlated with several studied clinicopathological parameters.
 Results: Our results showed that SHH methylation levels are significantly present in normal tissues as compared to tumor tissues.
 Conclusion: We conclude SHH promoter is hypomethylated in colorectal cancers which may have an effect driving normal colon and rectum cells to cancer.
 Bangladesh Journal of Medical Science Vol. 23 No. 01 January’24 Page : 200-206

Performance of Azolla pinnata fern protein hydrolysates as an emulsifier and nutraceutical ingredient in an O/W emulsion system and their effect on human gut microbiota and mammalian cells.

This study investigated the dual potential of Azolla pinnata fern protein hydrolysates (AFPHs) as functional and nutraceutical ingredients in an oil/water emulsion system. The AFPH-stabilised emulsion (AFPH-E) displayed a small and uniform droplet distribution and was stable to aggregation and creaming over a wide range of pH (5-8), salt concentrations ≤ 100 mM, and heat treatment ≤ 70 °C. Besides, the AFPH-E possessed and maintained strong biological activities, including antihypertensive, antidiabetic, and antioxidant, under different food processing conditions (pH 5-8; NaCl: 50-150 mM, and heat treatment: 30-100 °C). Following in vitro gastrointestinal digestion, the antihypertensive and antioxidant activities were unchanged, while a notable increase of 8% was observed for DPPH. However, the antidiabetic activities were partially reduced in the range of 5-11%. Notably, AFPH-E modulated the gut microbiota and short-chain fatty acids (SCFAs), promoting the growth of beneficial bacteria, particularly Bifidobacteria and Lactobacilli, along with increased SCFA acetate, propionate, and butyrate. Also, AFPH-E up to 10 mg mL-1 did not affect the proliferation of the normal colon cells. In the current work, AFPH demonstrated dual functionality as a plant-based emulsifier with strong biological activities in an oil/water emulsion system and promoted healthy changes in the human gut microbiota.

Transcriptome profiling of human col\onic cells exposed to the gut pathobiont Streptococcus gallolyticus subsp. gallolyticus.

Streptococcus gallolyticus sp. gallolyticus (SGG) is a gut pathobiont involved in the development of colorectal cancer (CRC). To decipher SGG contribution in tumor initiation and/or acceleration respectively, a global transcriptome was performed in human normal colonic cells (FHC) and in human tumoral colonic cells (HT29). To identify SGG-specific alterations, we chose the phylogenetically closest relative, Streptococcus gallolyticus subsp. macedonicus (SGM) as control bacterium. We show that SGM, a bacterium generally considered as safe, did not induce any transcriptional changes on the two human colonic cells. The transcriptional reprogramming induced by SGG in normal FHC and tumoral HT29 cells was significantly different, although most of the genes up- and down-regulated were associated with cancer disease. Top up-regulated genes related to cancer were: (i) IL-20, CLK1, SORBS2, ERG1, PIM1, SNORD3A for normal FHC cells and (ii) TSLP, BHLHA15, LAMP3, ZNF27B, KRT17, ATF3 for cancerous HT29 cells. The total number of altered genes were much higher in cancerous than in normal colonic cells (2,090 vs 128 genes being affected, respectively). Gene set enrichment analysis reveals that SGG-induced strong ER- (endoplasmic reticulum) stress and UPR- (unfolded protein response) activation in colonic epithelial cells. Our results suggest that SGG induces a pro-tumoral shift in human colonic cells particularly in transformed cells potentially accelerating tumor development in the colon.

Long non-coding RNA linc00659 promotes tumour progression by regulating FZD6/Wnt/β-catenin signalling pathway in colorectal cancer via m6A reader IGF2BP1.

Abnormal N6-methyladenosine (m6A) modification has become a driving factor in tumour development and progression. The linc00659 is abnormally highly expressed in digestive tract tumours and promotes cancer progression, but there is little research on the mechanism of linc00659 and m6A. The expression of linc00659 in colorectal cancer (CRC) tissues and cells was assessed by a quantitative real-time PCR. The proliferative capacity of CRC cells was determined by colony formation, Cell Counting Kit-8 and 5-ethynyl-2 deoxyuridine assays, and the migratory capacity of CRC was determined by wound healing and transwell assays and tube formation. In vivo, a xenograft tumour model was used to detect the effect of linc00659 on tumour growth. The Wnt/β-catenin signalling pathway and related protein expression levels were measured by western blotting. The binding of linc00659 to insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) was assessed by RNA pull-down and an immunoprecipitation assay. The effect of IGF2BP1 on FZD6 was detected by an RNA stability assay. The expression of linc00659 was abnormally elevated in CRC tissues and cells compared to normal colonic tissues and cells. We confirm that linc00659 promotes the growth of CRC cells both in vivo and in vitro. Mechanistically, linc00659 binds to IGF2BP1 and specifically enhances its activity to stabilize the target gene FZD6. Therefore, linc00659 and IGF2BP1 activate the Wnt/β-catenin signalling pathway, promoting cell proliferation in CRC. Our results show that linc00659 and IGF2BP1 cooperate to promote the stability of the target FZD6 mRNA, thereby facilitating CRC progression, which may represent a potential diagnostic, prognostic and therapeutic target for CRC.

The New Role of HNF1A-NAS1/miR-214/INHBA Signaling Axis in Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cancer and one of the leading causes of death worldwide. Seriously threatens human life and health. Previous studies have identified that inhibin βA (INHBA) could induce tumorgenesis and progression of CRC through the regulation of the TGF-β/Smad signal axis. The abnormal expression of INHBA is related to the poor prognosis of patients. The aim of this study was to identify the molecular mechanism of HNF1A-AS1 and miR-214 regulating INHBA and carcinogenesis through bioinformatics combined with experiments. The expression of HNF1A-AS1, miRNA-214-5p, INHBA in pan-cancer and CRC were investigated in the Cancer Genome Atlas (TCGA). The correlation between HNF1A-AS1 and immune-related genes or miRNAs was explored via the Gene Expression Profiling Interactive Analysis (GEPIA) and volcano plots, respectively. The association between HNF1A-AS1 and differentially expressed miRNAs was constructed by TargetScan. The miRDB, miRWalk, and TargetScan databases were utilized to predict the target genes of hsa-miR-214. The expression of INHBA in tissues and cell lines of CRC was examined by RT-qPCR and western blot assay. The INHBA and HNF1A-AS1 expressions were increased in Colon adenocarcinoma (COAD) and Rectum adenocarcinoma (READ) of the TCGA database. Hsa-miR-214 was relatively less expressed in CRC tissues compared with para-cancer tissues. The expression of HNF1A-AS1 was negatively correlated with hsa-miR-214. INHBA was one of the target genes of hsa-miR-214 based on miRDB, miRWalk, and TargetScan databases. The specific binding sites of INHBA-3'UTR and miR-214-5p were identified by starBase. The expression level of INHBA was positively correlated with the T stage of tumor and negatively correlated with overall survival (OS) and disease-free survival (DFS) in CRC patients. The results of RT-qPCR and western blot indicated that the expression of INHBA in tissues and cell lines in CRC was higher than those in para-carcinoma tissues and normal colon cell lines, respectively. These findings suggested that HNF1A-AS1 and miRNA-214-5p were key upstream non-coding RNAs of INHBA. The HNF1A-AS1/miR-214/INHBA signal axis plays a significant role in the tumorgenesis and progression of CRC. By interfering with HNF1A-AS1 and INHBA genes on HT29 and SW480 cells, it was found that HNF1A-AS1 and INHBA genes may be important target genes in CRC.

Ethyl acetate fraction of Osmanthus fragrans var. aurantiacus and its triterpenoids suppress proliferation and survival of colorectal cancer cells by inhibiting NF-κB and COX2

Ethnopharmacological relevanceColorectal cancer (CRC) remains a significant global health concern, and targeting inflammation has emerged as a promising approach for its prevention and treatment. Medicinal plants and phytochemicals have garnered attention for their potential efficacy against inflammation with minimal toxicity. Osmanthus fragrans var. aurantiacus Makino (O. fragrans) has a history of traditional use in Korea and China in treating various inflammation-related conditions, but its potential use for CRC has not been uncovered. Aim of the studyThis study aims to explore the potential anti-proliferative and pro-apoptotic properties of O. fragrans, focusing on its impact on CRC treatment. By investigating O. fragrans, we aim to uncover its anti-proliferative and apoptotic effects in human CRC cells, potentially paving the way for effective and well-tolerated therapeutic strategies for CRC patients. Materials and methodsEthanol (EtOH) extracts of O. fragrans leaf and flower, along with specific fractions (n-hexane, ethyl acetate (EtOAc), n-butanol, and the aqueous residue) were evaluated for their anti-proliferative effects in human CRC cells using MTT assays, and compared to normal colon cells. Mechanistic insights and chemical profiling were obtained through flow cytometry, colorimetric assays, western blotting, and molecular docking, and high-performance liquid chromatography (HPLC) system. ResultsBoth flower and leaf EtOH extracts of O. fragrans exhibited significant anti-proliferative effects in human CRC cells, with the leaf extract demonstrating higher potency. The EtOAc fraction from the leaf extract displayed the strongest anti-CRC cell proliferative effects while no cytotoxic effects in normal colon cells. Chemical profiling of these fractions identified triterpenoids as significant components in the EtOAc fractions. The leaf EtOAc fraction caused cell cycle arrest and apoptosis, accompanied by elevating intracellular reactive oxygen species and mitochondrial dysfunction in CRC cells. Additionally, it inhibited NF-κB and ERK1/2 signaling, leading to reduced COX2 expression. Notably, two triterpenoids isolated from the leaf EtOAc fraction, maslinic acid and corosolic acid, displayed potent anti-cancer activity in CRC cells without affecting normal colon cells. Corosolic acid exhibited a strong binding affinity to COX2 and reduced its expression, supporting its role in the anti-inflammatory and anti-cancer effects. ConclusionsOur findings suggest that O. fragrans, particularly its triterpenoid-rich EtOAc fraction, holds promise as a novel therapeutic agent for CRC prevention and therapy. These results provide valuable insights into the potential application of O. fragrans and its bioactive compounds in combating CRC.

High expression of ANKRD6 is an indicator for poor prognosis of colon adenocarcinoma

To explore the expression of ANKRD6 in colon adenocarcinoma (COAD) and its implications for prognosis and treatment of COAD. We investigated the differential expression of ANKRD6 in 20 pairs of COAD and adjacent tissues using immunohistochemistry and in colon cancer cell lines and normal colon cells using real-time quantitative PCR. Gene expression matrices, immune infiltration data, and clinical information of 521 COAD patients and 41 normal tissues were obtained from the TCGA database for analyzing the association of ANKRD6 expression with clinicopathological features, gene set enrichment, and immune infiltration in COAD using R software. The prognostic factors of COAD were evaluated using univariate and multivariate Cox regression and Kaplan-Meier analyses, and a prognostic nomogram containing ANKRD6 was constructed. Western blotting, CCK-8 assay, scratch assay and Transwell assay were performed to assess the effects of ANKRD6 knockdown on biological activities of COAD cells. ANKRD6 was highly expressed in COAD cell lines and upregulated in COAD tissues with higher malignancy (P<0.05). Increased ANKRD6 expression was significantly correlated with decreased overall survival and disease-free survival of COAD patients (P<0.05). Univariate and multivariate Cox regression analyses indicated that ANKRD6 expression was significantly correlated with the overall survival rate of COAD patients (P<0.05). The constructed prognostic nomogram showed a C-index value of 0.739 and effectively predicted the 1-, 3-, and 5-year survival of the patients. ANKRD6 expression was positively correlated with immune infiltration of macrophages (P<0.05) and negatively correlated with Th17 cells and other immune cell infiltration (P<0.05). Bioinformatic analysis suggested that ANKRD6 was mainly enriched in Wnt, vascular endothelial growth factor receptor, and calcium signaling pathways in COAD. In cultured COAD cells, ANKRD6 knockdown significantly suppressed cell proliferation, migration, and invasion (P<0.05). High ANKRD6 expression is an indicator for poor prognosis of COAD, and ANKRD6 may serve as a potential biomarker as well as a therapeutic target for diagnosis and treatment of COAD.

Screening and validation of the optimal panel of reference genes in colonic epithelium and relative cancer cell lines

Real-time quantitative polymerase chain reaction (RT-qPCR) is the most common method to determine mRNA expression, and Minimum Information for Publication of RT-qPCR Experiments (MIQE) proposes that a panel of reference genes for RT-qPCR is conducive to obtaining accurate results. This study aimed to screen and verify the optimal panel of reference genes in colorectal cancer (CRC) and normal colonic cell lines. In the study, eight candidate reference genes (GAPDH, ACTB, 18S, PPIA, B2M, SDHA, GUSB, and YWHAZ) were selected for RT-qPCR to detect their expression in NCM460, HT29, HCT116, SW480, SW620, DLD-1, LOVO and RKO cell lines. The stability of reference genes and the optimal panel were evaluated by geNorm, NormFinder, and BestKeeper software. As results, the expression levels of candidate reference genes differed in the colonic epithelial cell lines, and the number of optimal panel of reference genes is two. B2M and YWHAZ were the two most stable reference genes for NCM460, HCT116, SW620, LOVO, and RKO cell lines, while only one of B2M and YWHAZ was most stable in HT29 and SW480 cells. In DLD-1 cells, the stability of B2M and YWHAZ ranked 3rd and 6th, PPIA and GUSB were the most stable two. Furthermore, the YWHZA + B2M performed smaller intragroup differences than other panel or single reference gene. In conclusion, this study indicates the optimal panel of reference genes is YWHZA + B2M for the NCM460, HCT116, SW620, LOVO, RKO, SW480, and HT29 cell lines, but it is PPIA + GUSB in DLD-1 cell lines.

METTL3/IGF2BP2 axis affects the progression of colorectal cancer by regulating m6A modification of STAG3

Colorectal cancer (CRC) is among the commonest malignant tumors of humans. Existing evidence has linked the poor prognosis of CRC with high expression of stromal antigen 3 (STAG3), but, the exact biological effect of STAG3 in CRC is still unclear. The aim of this research is to reveal the biological function and molecular mechanism of STAG3 in CRC. To investigate the differential expression of STAG3 in CRC tissues and cell lines compared to normal colon tissues and cell lines, Western blot (WB) and quantitative real-time PCR (qRT-PCR) techniques were utilized. STAG3 N6-methyladenosine (m6A) modification level were identified using m6A RNA immunoprecipitation (MeRIP). Additionally, the functional roles of methyltransferase-like protein 3 (METTL3) and insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) in CRC were explored by manipulating their levels via knockdown or overexpression. Cell proliferation was evaluated through Cell Counting Kit 8 (CCK-8) and clone formation experiments, while cell migration was assessed through wound healing experiments. Furthermore, cell apoptosis was detected using flow cytometry, and the protein expressions associated with proliferation and apoptosis were detected using WB. To identify the specific binding of target genes, RIP and pull-down assays were employed. Finally, the biological function of STAG3 in vivo was investigated through a xenotransplantation mouse tumor model. In CRC tissues and cell lines, STAG3 was up-regulated and accompanied by m6A methylation. Additionally, the expression of METTL3 was found to be upregulated in CRC tissues. Knocking down METTL3 resulted in a decrease in both the m6A level and protein expression of STAG3, inhibited cell proliferation and migration while promoting apoptosis, which were restored through STAG3 overexpression. Furthermore, online prediction indicated the interaction between STAG3 mRNA and IGF2BP2 protein, which was further verified by RIP experiments. IGF2BP2 downregulation led to decreased STAG3 protein expression, cell proliferation, and migration, but increased apoptosis. However, these impacts were reversed by STAG3 overexpression. Finally, subcutaneous tumor experiments conducted in nude mice also confirmed that METTL3 regulated CRC progression through STAG3 in vivo. The METTL3/IGF2BP2/STAG3 axis affects CRC progression in an m6A modification-dependent manner. This may guide targeted therapy in CRC patients.