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Colon cancer is a significant health problem worldwide as it is one of the most common and deadliest cancers. The standard approach for the treatment of colon cancer is 5-fluorouracil (5-FU) based chemotherapy, which is limited by the development of resistance to this drug. Therefore, our study aimed to establish 5-FU resistance in SW-480 and HT-29 colon cancer cells and to precisely determine the molecular mechanisms and biomarkers that contribute to its development, both after short-term exposure and in cells with already developed resistance (SW-480-5FUR and HT-29-5FUR). The expression of various molecules involved in the different mechanisms of resistance development was monitored at the gene (qPCR) and protein (immunocytochemistry) levels. Based on the obtained results, alterations in the 5-FU anabolic pathway, biotransformation, drug efflux, mismatch repair, and apoptosis process together contributed to the development of 5-FU resistance in SW-480 and HT-29 colon cancer cells. In addition, UMPS, ABCC1, ABCC5, and MLH1, as well as the disturbed ratio of pro-apoptotic BAX and anti-apoptotic BCL2, should be taken into consideration as potential targets for the discovery of 5-FU resistance-related biomarkers in colon cancer cells. We suggest that future investigations focus on further validation of these findings by additional in vitro and in vivo testing, which is a limitation of our study.

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Background: Tumor cell inhibition is a pivotal focus in anti-cancer research, and extensive investigations have been conducted regarding the role of p53. Numerous studies have highlighted its close association with reactive oxygen species (ROS). However, the precise impact of the antioxidant glutathione (GSH) in this context remains inadequately elucidated. Here, we will elucidate the anti-cancer mechanisms mediated by p53 following treatment with GSH. Methods: In this study, we employed a p53 gene knockout approach in SW480 colorectal cells and conducted comprehensive analyses of 20 amino acids and proteomics using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Results: These analyses unveiled profound alterations in amino acids and proteins triggered by GSH treatment, shedding light on novel phenomena and delineating the intricate interplay between GSH and cellular proteins. The deletion of the p53 gene exerts a profound influence on tumor cell proliferation. Moreover, tumor cell proliferation is significantly affected by elevated GSH levels. Importantly, in the absence of the p53 gene, cells exhibit heightened sensitivity to GSH, leading to inhibited cell growth. The combined therapeutic approach involving GSH and p53 gene deletion expedites the demise of tumor cells. It is noteworthy that this treatment leads to a marked decline in amino acid metabolism, particularly affecting the down-regulation of methionine (Met) and phenylalanine (Phe) amino acids. Among the 41 proteins displaying significant changes, 8 exhibit consistent alterations, with 5 experiencing decreased levels and 3 demonstrating increased quantities. These proteins primarily participate in crucial cellular metabolic processes and immune functions. Conclusions: In conclusion, the concurrent administration of GSH treatment and p53 gene deletion triggers substantial modifications in the amino acid and protein metabolism of tumor cells, primarily characterized by down-regulation. This, in turn, compromises cell metabolic activity and immune function, ultimately culminating in the demise of tumor cells. These newfound insights hold promising implications and could pave the way for the development of straightforward and efficacious anti-cancer treatments.

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A growing number of studies have suggested the involvement of long non-coding RNAs as the key players in not just the initiation and progression of the tumor microenvironment, but also in chemotherapy tolerance. In the present study, generated 5-FU-resistant SW480/DR cells were analyzed via cDNA microarray for its aberrant lncRNAs and mRNAs expression in comparison with the 5-FU-susceptible SW480/DS cells. Among the 126 lncRNAs described, lncRNAs GNAS-AS1, MIR205HG, and LOC102723721 have been identified to be significantly upregulated, while lncRNs lnc-RP11-597K23.2.1-2, LOC100507639, and CCDC144NL-AS1 have been found to be significantly downregulated. In the meantime, bioinformatic analysis through gene ontology studies of aberrantly expressed mRNAs revealed "regulated exocytosis", among others, as the biological process most impacted in SW480/DR cells. To investigate, exosome purification was then carried out and its characterization were validated via transmission electron microscopy and nanoparticle tracking analysis. Interestingly, it was determined that the 5-FU-resistant SW480/DR cells secretes significantly higher concentration of extracellular vesicles, particularly, exosomes when compared to the 5-FU-susceptible SW480/DS cells. Based on the lncRNA-mRNA interaction network analysis generated, lncRNA GNAS-AS1 and MIR205HG have been identified to be potentially involved in the incidence of 5-FU resistance in SW480 colon cancer cells through promoting increased release of exosomes into the intercellular matrix. Our study hopes not only to provide insights on the list of involved candidate lncRNAs, but also to elucidate the role exosomes play in the initiation and development of 5-FU chemotherapy resistance in colon cancer cells.

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Background: There are some problems in the clinical diagnosis of colorectal cancer (CRC), such as the difficulty in saving samples, so it is the most popular research work to develop a diagnostic index and method that is easy to obtain, convenient to save and stable. G-quadruplex (G4) is a unique structure found in DNA and it plays a crucial biological role in tumor formation. G4 is derived from DNA with good stability, and the DNA of serum samples is easy to obtain. Therefore, G4 has the potential as an ideal marker for CRC diagnosis. However, it has not received more attention. Methods: Through bioinformatics-based G4 mutation prediction in the genome, we discovered that the G4 quantity in SW480 cells was lower than that of the reference gene. However, it was unclear how the G4 quantity changed in the actual samples. We detected the G4 content by fluorescence in cells and human serum samples. Results: G4 content was significantly higher than that in NCM480 (P<0.001). To further explore the relationship between tumorigenesis and G4, we knocked out the TP53 gene in SW480 cells and found that the G4 content decreased significantly (64%) (P<0.001). The difference in G4 content is a key factor in distinguishing between normal and tumor cells. Furthermore, we detected G4 in serum samples from 27 healthy individuals and 27 patients with CRC and found that G4 was significantly increased in those with CRC (P<0.001) by 1.94 fold. We also evaluated the G4 model using receiver operating characteristic (ROC), with an area under the curve of 0.91, and found it to have excellent specificity and sensitivity. Conclusions: The increased G4 is an important characteristic in patients with CRC and has clinical application value as a novel biomarker. The relationship between G4 and TP53 regulation may be a potential target for future cancer studies, and as attention to this area of research increases, the underlying mechanisms may be better understood, potentially benefiting clinical cancer treatment.

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BACKGROUND: Although extensive efforts have been made to improve the treatment of colorectal cancer (CRC) patients, the prognosis for these patients remains poor. A wide range of anti-cancer agents has been applied to ameliorate the clinical management of CRC patients; however, drug resistance develops in nearly all patients. Based on the prominent role of PI3K/AKT signaling in the development of CRC and current interest in the application of PI3K inhibitors, we aimed to disclose the exact mechanism underlying the efficacy of BKM120, a well-known pan-class I PI3K inhibitor, in CRC-derived SW480 cells. MATERIALS AND METHODS: The effects of BKM120 on SW480 cells were studied using MTT assay, cell cycle assay, Annexin V/PI apoptosis tests, and scratch assay. In the next step, qRT-PCR was used to investigate the underlying molecular mechanisms by which the PI3K inhibitor could suppress the survival of SW480 cells. RESULT: The results of the MTT assay showed that BKM120 could decrease the metabolic activity of SW480 cells in a concentration and time-dependent manner. Investigating the exact mechanism of BKM120 showed that this PI3K inhibitor induces its anti-survival effects through a G2/M cell cycle arrest and apoptosis-mediated cell death. Moreover, the scratch assay demonstrated that PI3K inhibition led to the inhibition of cancer invasion and inhibition of PI3K/AKT signaling remarkably sensitized SW480 cells to Cisplatin. CONCLUSION: Based on our results, inhibition of PI3K/AKT signaling can be a promising approach, either as a single modality or in combination with Cisplatin. However, further clinical studies should be performed to improve our understanding.

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Iron overload occurs due to excessive iron intake compared to the body's demand, leading to iron deposition and impairment of multiple organ functions. Our previous study demonstrated that chronic oral administration of ferric citrate (FC) caused colonic inflammatory injury. However, the precise mechanism underlying this inflammatory response remains unclear. The current study aims to investigate the mechanism by which iron overload induced by FC exposure leads to colonic inflammation. To accomplish this, mice were orally exposed to three different concentrations of FC (71 mg/kg/bw (L), 143 mg/kg/bw (M) and 286 mg/kg/bw (H)) for continuous 16 weeks, with the control group receiving ultrapure water (C). Exposure to FC caused disturbances in the excretory system, altered colonic flora alpha diversity, and enriched pathogenic bacteria, such as Mucispirillum, Helicobacter, Desulfovibrio, and Shigella. These changes led to structural disorders of the colonic flora and an inflammatory response phenotype characterized by inflammatory cells infiltration, atrophy of intestinal glands, and irregular thickening of the intestinal wall. Mechanistic studies revealed that FC-exposure activated the NF-κB signaling pathway by up-regulating TLR4, MyD88, and NF-κB mRNA levels and protein expression. This activation resulted in increased production of pro-inflammatory cytokines, further contributing to the colonic inflammation. Additionally, in vitro experiments in SW480 cells confirmed the activation of NF-κB signaling pathway by FC exposure, consistent with the in vivo findings. The significance of this study lies in its elucidation of the mechanism by which iron overload caused by FC exposure leads to colonic inflammation. By identifying the role of pathogenic bacteria and the NF-κB signaling pathway, this study could potentially offer a crucial theoretical foundation for the research on iron overload, as well as provide valuable insights for clinical iron supplementation.

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BACKGROUND: The use of nanomaterials in cancer diagnosis and treatment has received considerable interest. Preparation of nanoscale complex molecules could be considered to improve the efficacy and minimize toxicity of the product. This work aimed to biosynthesize BiFe2O4@Ag nanocomposite using the Chlorella vulgaris extract and its cytotoxic effect on colon cancer cell line. METHODS: The physicochemical properties of the bioengineered BiFe2O4 @Ag were investigated by Transmission Electron Microscopy (TEM), Field Emission Scanning Electron Microscopy (FE-SEM), Zeta potential, Dynamic Light Scattering (DLS), Fourier Transform Infrared Spectroscopy (FT-IR), Energy Dispersive X-ray Spectroscopy (EDX), Vibrating-sample Magnetometer (VSM) and X-ray Diffraction Analysis (XRD). The cytotoxic potential of BiFe2O4 @Ag was evaluated by MTT assay against SW480 colon cancer cell line. The expression levels of apoptotic genes including BAX, BCL2 and CASP8 were determined by Real-time PCR. The rate of apoptosis and necrosis of the cancer cells as well as the cell cycle analysis were evaluated by flow cytometry. RESULTS: Physicochemical assays indicated the nanoscale synthesis (10-70 nm) and functionalization of BiFe2O4 nanoparticles by Ag atoms. The VSM analysis revealed the magnetism of BiFe2O4 @Ag nanocomposite. According to the MTT assay, colon cancer cells (SW480) were considerably more sensitive to BiFe2O4 @Ag nanocomposite than normal cells. Apoptotic cell percentage increased from 1.93% to 73.66%, after exposure to the nanocomposite. Cell cycle analysis confirmed an increase in the number of the cells in subG1 and G0/G1 phases among nanocomposite treated cells. Moreover, treating the colon cancer cells with BiFe2O4 @Ag caused an increase in the expression of CASP8, BAX, and BCL2 genes by 3.1, 2.6, and 1.2 folds, respectively. Moreover, activity of Caspase-3 protein increased by 2.4 folds and apoptotic morphological changes appeared which confirms that exposure to the nanocomposite induces extrinsic pathway of apoptosis in colon cancer cells. CONCLUSION: The considerable anticancer potential of the synthesized BiFe2O4 @Ag nanocomposite seems to be related to the induction of oxidative stress which leads to inhibit cell cycle progression and cell proliferation. This study reveals that the BiFe2O4 @Ag is a potent compound to be used in biomedical fields.

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Ferula gummosa Boiss. is a well-known Iranian endemic plant that grows in the north and northeast regions of Iran. In Iranian traditional medicine, its gum is utilized to treat inflammation, pain, and infections of the gastrointestinal system. However, no studies have been conducted to investigate the anticancer potential of its gum against colorectal cancer cells. This study aimed to identify the chemical components of the gum of F. gummosa and investigate its effects on SW-480 cells. The experiments included MTT, clonogenic, micronucleus formation, acridine orange/ethidium bromide stain, DNA degradation, caspase 3/7 activity assay, and in vitro wound-healing experiment and investigating the expression of BAX, BCL2, MTOR, and PTEN genes. Chemical analysis using GC/MS identified 102 compounds. The gum had a significant cytotoxic effect on SW-480 cells, with an IC50 value of 1.8 µg/ml for 48 hours. The gum induced apoptosis. Microscopic observations revealed a decrease in cell proliferation, as evidenced by nuclear condensation, increased micronucleus formation, and inhibition of colony formation. Additionally, the gum suppressed cell migration, induced the expression of PTEN and BAX, and down-regulated MTOR and BCL2 genes. These findings suggest that Ferula gummosa has strong cytotoxic properties and warrants further investigation.

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OBJECTIVES: The invasive screening methods and the late stage diagnosis of colorectal carcinoma (CRC) are contributing for the devastative prognosis. The gradual shift of the disease pattern among younger generations requires the implementation of phytochemicals and traditional medicines. Arkeshwara rasa (AR) is a herb-mineral combination of Tamra bhasma/incinerated copper ashes and Dwigun Kajjali/mercury sulphide levigated with Calotropis procera leaf juice, Plumbago zeylanica root decoction and the decoction of three myrobalans (Terminalia chebula, Terminalia bellerica, Emblica Officinalis decoction)/Triphala decoction. METHODS: The SW-480 cell line was checked for the cytotoxicity and the cell viability criteria with MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. The acridine orange/ethidium bromide (AO/EtBr) assay revealed the depth of apoptosis affected cells in the fluorescent images. The FTIR analysis exhibited the graphical spectrum of functional groups within the compound AR. RESULTS: The IC50 from the 10-7 to 10-3 concentrations against SW-480 cells was 40.4 µg/mL. The staining of AO/EtBr was performed to visualize live and dead cells and it is evident from the result that number of apoptotic cells increases at increasing concentration of AR. The single bond with stretch vibrations of O-H and N-H are more concentrated in the 2,500-3,200 cm-1 and 3,700-4,000 cm-1 of the spectra whereas, the finger print region carries the O-H and S=O type peaks. CONCLUSIONS: The AR shows strong cyto-toxicity against the SW-480 cells by inducing apoptosis. It also modulates cellular metabolism with the involvement of functional groups which antagonizes the strong acids. Moreover, these effects need to be analyzed further based in the in vivo and various in vitro models.

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@#[摘 要] 目的：探讨异莲心碱（Iso）通过PI3K/Akt/mTOR信号通路对结肠癌SW480细胞增殖、凋亡和自噬的影响。方法：用10、20和40 μmol/L的Iso处理结肠癌SW480细胞，CCK-8法、流式细胞术和WB法分别检测Iso对细胞增殖活力、凋亡和自噬相关蛋白LC3Ⅰ、LC3Ⅱ、p62表达的影响。然后，用20 μmol/L的Iso和25 μmol/L的PI3K激活剂740 Y-P分别处理SW480细胞，将细胞分为对照组、740 Y-P组、Iso组和Iso+740 Y-P组，流式细胞术、WB法检测Iso和740 Y-P对各组细胞凋亡及细胞中LC3Ⅰ、LC3Ⅱ、p62、PI3K、p-PI3K、 mTOR和p-mTOR蛋白表达的影响。结果：10、20和40 μmol/L的Iso处理后，SW480细胞增殖活力均显著下降（均P<0.05），细胞凋亡率均显著升高（均P<0.05），LC3Ⅱ/LC3Ⅰ表达均显著上调（均P<0.05），p26蛋白表达显著下调（P<0.05）。Iso和740 Y-P处理后，与对照组相比，740 Y-P组细胞凋亡率、LC3Ⅱ/LC3Ⅰ表达均显著下降（均P<0.05），p26、p-PI3K/PI3K和p-mTOR/mTOR表达均显著升高（均P<0.05）；Iso组细胞凋亡率、LC3Ⅱ/LC3Ⅰ表达升高（均P<0.05），p26、p-PI3K/PI3K和p-mTOR/mTOR表达均显著下降（均P<0.05）；与740 Y-P组相比，Iso+740 Y-P组细胞凋亡率、LC3Ⅱ/LC3Ⅰ表达升高（P<0.05），p26、p-PI3K/PI3K和p-mTOR/mTOR表达均显著下降（均P<0.05）；与Iso组相比，Iso+740 Y-P组细胞凋亡率、LC3Ⅱ/LC3Ⅰ表达下降（均P<0.05），p26、p-PI3K/PI3K和p-mTOR/mTOR表达均显著升高（均P<0.05）。结论：Iso通过抑制PI3K/Akt/mTOR信号通路抑制结肠癌SW480细胞增殖并诱导细胞凋亡和自噬。

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To overcome the limitations of in vitro two-dimensional (2D) cancer models in mimicking the complexities of the native tumor milieu, three-dimensional (3D) engineered cancer models using biomimetic materials have been introduced to more closely recapitulate the key attributes of the tumor microenvironment. Specifically, for colorectal cancer (CRC), a few studies have developed 3D engineered tumor models to investigate cell-cell interactions or efficacy of anti-cancer drugs. However, recapitulation of CRC cell line phenotypic differences within a 3D engineered matrix has not been systematically investigated. Here, we developed an in vitro 3D engineered CRC (3D-eCRC) tissue model using the natural-synthetic hybrid biomaterial PEG-fibrinogen and three CRC cell lines, HCT 116, HT-29, and SW480. To better recapitulate native tumor conditions, our 3D-eCRC model supported higher cell density encapsulation (20 × 106 cells/mL) and enabled longer term maintenance (29 days) as compared to previously reported in vitro CRC models. The 3D-eCRCs formed using each cell line demonstrated line-dependent differences in cellular and tissue properties, including cellular growth and morphology, cell subpopulations, cell size, cell granularity, migration patterns, tissue growth, gene expression, and tissue stiffness. Importantly, these differences were found to be most prominent from Day 22 to Day 29, thereby indicating the importance of long-term culture of engineered CRC tissues for recapitulation and investigation of mechanistic differences and drug response. Our 3D-eCRC tissue model showed high potential for supporting future in vitro comparative studies of disease progression, metastatic mechanisms, and anti-cancer drug candidate response in a CRC cell line-dependent manner.

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A new amine, zeaamine (1), along with nine known compounds (2-10), were isolated from the roots of Zea mays. Among these, compound 2 was first isolated from this plant, and compound 3 was first isolated from the roots. In the current investigation, the cytotoxicity against CT26 and SW480 cells of the compounds were evaluated. Zeaamine (1) exhibited moderately affected CT26 and SW480 cells with IC50 values of 17.91 and 10.21 µM.

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Parkinson's disease (PD) is a globally common progressive neurodegenerative disease resulting from the loss of dopaminergic neurons in the brain. Increased α-synuclein (α-syn) is associated with the degeneration of dopaminergic neurons and non-motor symptoms like gastrointestinal disorders. In this study, we investigated the association between serum/glucocorticoid-related kinase 1 (SGK1) and α-syn in the colon of a PD mouse model. SGK1 and α-syn expression patterns were opposite in the surrounding colon tissue, with decreased SGK1 expression and increased α-syn expression in the PD group. Immunofluorescence analyses revealed the colocation of SGK1 and α-syn; the PD group demonstrated weaker SGK1 expression and stronger α-syn expression than the control group. Immunoblotting analysis showed that Na+/K+ pump ATPase α1 expression levels were significantly increased in the PD group. In SW480 cells with SGK1 knockdown using SGK1 siRNA, decreasing SGK1 levels corresponded with significant increases in the expression levels of α-syn and ATPase α1. These results suggest that SGK1 significantly regulates Na+/K+ pump ATPase, influencing the relationship between electrolyte balance and fecal formation in the PD mouse model. Gastrointestinal disorders are some of the major prodromal symptoms of PD. Therefore, modulating SGK1 expression could be an important strategy for controlling PD.

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This study is aimed to investigate the effects of Hypericum perforatum olive oil extract on the cytotoxic and metastatic properties of human colorectal cancer cells and human bone marrow-derived mesenchymal stem cells. In addition, ALDH3A1 and Vimentin expressions were evaluated by qRT-PCR and western blot analysis. Total phenolic and flavonoid contents and antioxidant activity of methanol extracts prepared with oil enrichment were measured using spectrophotometry-based methods. The cytotoxic effects of the extracts on SW-480 and bone marrow-derived mesenchymal stem cells were evaluated by MTT assay, resulting in IC50 values of 4.8 mg/ml and 4.9 mg/ml, respectively. It was determined that cell migration and colony formation were significantly reduced at the IC50 values determined for SW-480 and human bone marrow-derived mesenchymal stem cells.

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Colorectal cancer (CRC) is a common and highly malignant neoplasm, ranking as the fourth most frequent cause of cancer-related deaths worldwide. Recently, non-human oncolytic viruses such as Peste des petits ruminants virus (PPRV) are considered as a potent candidate in the viral therapy of cancer. In the current study, the apoptotic effects of matrix (M) protein of PPRV was investigated on SW480 CRC cells. The M gene was cloned into the pcDNA™3.1/Hygro(+) expression vector and transfected into the cancer cells. The cytotoxic effects of the M protein on SW480 cells were confirmed using MTT assay. Furthermore, flow cytometry results showed that the M protein induces apoptosis in 91 % of CRC cells. Interestingly, the expression of the M gene in SW480 cells led to the up-regulation of genes including Bax, p53, and Caspase-9, as well as an increase in the Bax/Bcl-2 ratio. By using bioinformatics modeling, we hypothesized that the M protein could interact with Bax factor through its BH3-like motif and could further activate the intrinsic apoptosis pathway. Ultimately, this study provided the first evidence of the pro-apoptotic activity of PPRV M protein indicating its possible development as a promising novel anti-cancer agent.

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Early cancer screening enables timely detection of carcinogenesis, and aids in prompt clinical intervention. Herein, we report on the development of a simple, sensitive, and rapid fluorometric assay based on the aptamer probe (aptamer beacon probe, ABP) for monitoring the energy-demand biomarker adenosine triphosphate (ATP), an essential energy source that is released into the tumor microenvironment. Its level plays a significant role in risk assessment of malignancies. The operation of the ABP for ATP was examined using solutions of ATP and other nucleotides (UTP, GTP, CTP), followed by monitoring of ATP production in SW480 cancer cells. Then, the effect of a glycolysis inhibitor, 2-deoxyglucose (2-DG), on SW480 cells was investigated. The stability of predominant ABP conformations in the temperature range of 23-91 °C and the effects of temperature on ABP interactions with ATP, UTP, GTP, and CTP were evaluated based on quenching efficiencies (QE) and Stern-Volmer constants (KSV). The optimized temperature for best selectivity of ABP toward ATP was 40 °C (KSV = 1093 M-1, QE = 42%). We have found that the inhibition of glycolysis in SW480 cancer cells by 2-deoxyglucose resulted in lowering of ATP production by 31.7%. Therefore, monitoring and modulation of ATP concentration may aid in future cancer treatment.

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PURPOSE: Suppressor of mothers against decapentaplegic homolog 4 (SMAD family member 4, SMAD4) is involved in the adenoma-carcinoma pathway, leading to the development of colon cancer. The encoded protein is a key downstream signaling mediator in the TGFß pathway. This pathway has tumor-suppressor functions, including cell-cycle arrest and apoptosis. Its activation in late-stage cancer can promote tumorigenesis, including metastasis and chemoresistance. Most colorectal cancer patients receive chemotherapy based on 5-FU as an adjuvant treatment. However, the success of therapy is hampered by multidrug resistance by neoplastic cells. In colorectal cancer, resistance to 5-FU-based therapy is influenced by SMAD4 gene expression, as patients with decreased SMAD4 gene expression probably have a higher risk of developing 5-FU-induced resistance. The mechanism leading to the development of this phenomenon is not fully understood. Therefore, the present study assesses the possible influence of 5-FU on changes in the expression of the SMAD4 and TGFB1 genes. PATIENTS AND METHODS: The effect of 5-FU on the expression of SMAD4 and TGFB1 in colorectal cancer cells derived from the CACO-2, SW480 and SW620 cell lines was evaluated using real-time PCR. The cytotoxicity of 5-FU on colon cancer cells was assessed by the MTT method, and its effect on the induction of cell apoptosis and the initiation of DNA damage using a flow cytometer. RESULTS: Significant changes in the level of SMAD4 and TGFB1 gene expression were noted in the CACO-2, SW480 and SW620 cells treated with 5-FU at various concentrations during 24 h and 48 h exposure. The use of 5-FU at a concentration of 5 µmol/L resulted in a decrease in the expression of the SMAD4 gene in all cell lines at both exposure times, while the concentration of 100 µmol/L increased the expression of the SMAD4 gene in CACO-2 cells. The level of expression of the TGFB1 gene was higher for all cells treated with 5-FU at the highest concentrations, while the exposure time was extended to 48 h. CONCLUSION: The observed in vitro changes in CACO-2 cells caused by 5-FU may be of clinical relevance when choosing the drug concentration for treating patients with colorectal cancer. It is possible that 5-FU has a stronger effect on colorectal cancer cells at the higher concentrations. Low concentrations of 5-FU may not have a therapeutic effect and may also influence drug resistance in cancer cells. Higher concentrations and prolonged exposure time may affect SMAD4 gene expression, which may increase the effectiveness of therapy.

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BACKGROUND AND PURPOSE: Wnt binding to Frizzleds (FZD) is a crucial step that leads to the initiation of signalling cascades governing multiple processes during embryonic development, stem cell regulation and adult tissue homeostasis. Recent efforts have enabled us to shed light on Wnt-FZD pharmacology using overexpressed HEK293 cells. However, assessing ligand binding at endogenous receptor expression levels is important due to differential binding behaviour in a native environment. Here, we study FZD paralogue, FZD7 , and analyse its interactions with Wnt-3a in live CRISPR-Cas9-edited SW480 cells typifying colorectal cancer. EXPERIMENTAL APPROACH: SW480 cells were CRISPR-Cas9-edited to insert a HiBiT tag on the N-terminus of FZD7 , preserving the native signal peptide. These cells were used to study eGFP-Wnt-3a association with endogenous and overexpressed HiBiT-FZD7 using NanoBiT/bioluminescence resonance energy transfer (BRET) and NanoBiT to measure ligand binding and receptor internalization. KEY RESULTS: With this new assay the binding of eGFP-Wnt-3a to endogenous HiBiT-FZD7 was compared with overexpressed receptors. Receptor overexpression results in increased membrane dynamics, leading to an apparent decrease in binding on-rate and consequently in higher, up to 10 times, calculated Kd . Thus, measurements of binding affinities to FZD7 obtained in overexpressed cells are suboptimal compared with the measurements from endogenously expressing cells. CONCLUSIONS AND IMPLICATIONS: Binding affinity measurements in the overexpressing cells fail to replicate ligand binding affinities assessed in a (patho)physiologically relevant context where receptor expression is lower. Therefore, future studies on Wnt-FZD7 binding should be performed using receptors expressed under endogenous promotion.

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Cu nanoparticles (CuNPs) have various applications in biomedicine, owing to their unique properties. As the effect of CuNPs on the induction of oxidative stress and apoptosis in the human colorectal cancer cell line SW480 has not yet been studied, we investigated the toxicity and mechanism of action of these NPs in SW480 cells. MTT assay was performed to assess the effect of the particles on the viability of SW480 cells. The levels of oxidative stress were assessed after 24 h of treatment with CuNPs by evaluating the Reactive Oxygen Specious (ROS) production. The antioxidant enzyme activity was assessed using a colorimetric method. To investigate the effect of NPs on cellular apoptosis, Hoechst33258 staining was performed, and the expression of Bax, Bcl-2, and p53 was evaluated by qRT-PCR. The MTT assay results showed that CuNPs inhibited the viability of SW480 cells. Moreover, the increase in ROS production at all three concentrations (31, 68, and 100 µg/ml) was significant. It has been observed that CuNPs lead to increased expression of Bax and p53, and decreased expression of Bcl-2. Hoechst staining was performed to confirm apoptosis. In conclusion, the induction of apoptosis demonstrated the anticancer potential of the CuNPs.

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OBJECTIVE: In order to overcome the insolution and low bioavailability of the vitexin in vivo, ß-cyclodextrin-vitexin (ß-CD-vitexin) microspheres were prepared, and their effects on the proliferation of SW480 cells were observed. METHODS: Scanning electron microscopy, ultraviolet spectrum, Fourier transform infrared spectroscopy, and release rate analysis identified the formation of ß-CD-vitexin microspheres. MTT assay detected the effect of ß-CD-vitexin microspheres on tumor cell proliferation at 6, 12, 24, and 48 h. Fluorescence microscopy and flow cytometry were used to observe the effect of ß-CD-vitexin microspheres on the apoptosis of SW480 cells. The mRNA expression of the p53 gene was measured by qPCR. RESULTS: ß-CD-vitexin microspheres were successfully prepared. SW480 cell proliferation was inhibited by 0.1, 0.2, and 0.4 mg/mL of ß-CD-vitexin microspheres in a dose- and time-dependent manner, and the mechanism of proliferation inhibition was related to cell apoptosis caused by the upregulated expression of p53 gene. CONCLUSION: The preparation of ß-CD-vitexin sustained release microspheres is feasible, and ß-CDvitexin microspheres have potential anti-colorectal cancer value.

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