Short-chain fatty acids reprogram metabolic profiles with the induction of reactive oxygen species production in human colorectal adenocarcinoma cells.

Short-chain fatty acids (SCFAs) exhibit anticancer activity in cellular and animal models of colon cancer. Acetate, propionate, and butyrate are the three major SCFAs produced from dietary fiber by gut microbiota fermentation and have beneficial effects on human health. Most previous studies on the antitumor mechanisms of SCFAs have focused on specific metabolites or genes involved in antitumor pathways, such as reactive oxygen species (ROS) biosynthesis. In this study, we performed a systematic and unbiased analysis of the effects of acetate, propionate, and butyrate on ROS levels and metabolic and transcriptomic signatures at physiological concentrations in human colorectal adenocarcinoma cells. We observed significantly elevated levels of ROS in the treated cells. Furthermore, significantly regulated signatures were involved in overlapping pathways at metabolic and transcriptomic levels, including ROS response and metabolism, fatty acid transport and metabolism, glucose response and metabolism, mitochondrial transport and respiratory chain complex, one-carbon metabolism, amino acid transport and metabolism, and glutaminolysis, which are directly or indirectly linked to ROS production. Additionally, metabolic and transcriptomic regulation occurred in a SCFAs types-dependent manner, with an increasing degree from acetate to propionate and then to butyrate. This study provides a comprehensive analysis of how SCFAs induce ROS production and modulate metabolic and transcriptomic levels in colon cancer cells, which is vital for understanding the mechanisms of the effects of SCFAs on antitumor activity in colon cancer.

The Potential Inhibitory Role of Acetyl-L-Carnitine on Proliferation, Migration, and Gene Expression in HepG2 and HT29 Human Adenocarcinoma Cell Lines.

Malignancies of the liver and colon are the most prevalent forms of digestive system cancer globally. Chemotherapy, one of the most significant treatments, has severe side effects. Chemoprevention using natural or synthetic medications can potentially reduce cancer severity. Acetyl-L-carnitine (ALC) is an acetylated derivative of carnitine essential for intermediate metabolism in most tissues. This study aimed to investigate the effects of ALC on the proliferation, migration, and gene expression of human liver (HepG2) and colorectal (HT29) adenocarcinoma cell lines. The cell viability and half maximal inhibitory concentration of both cancer cell lines were determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Wound healing after treatment was assessed using a migration assay. Morphological changes were imaged using brightfield and fluorescence microscopy. Post treatment, apoptotic DNA was detected using a DNA fragmentation assay. The relative mRNA expressions of matrix metallopeptidase 9 (MMP9) and vascular endothelial growth factor (VEGF) were evaluated using RT-PCR. The results showed that ALC treatment affects the wound-healing ability of HepG2 and HT29 cell lines. Changes in nuclear morphology were detected under fluorescent microscopy. ALC also downregulates the expression levels of MMP9 and VEGF in HepG2 and HT29 cell lines. Our results indicate that the anticancer action of ALC is likely mediated by a decrease in adhesion, migration, and invasion.

Synthesis of selenophene-based chalcone analogs and assessment of their biological activity as anticancer agents.

Selenium is an essential micronutrient that is beneficial to human health. Selenium-containing drugs have been developed as antioxidants, anti-inflammatory, and anticancer agents. However, the synthesis of selenium-containing chalcones has not been fully explored. Therefore, we report the synthesis of novel selenophene-based chalcone analogs and reveal their biological activities as anticancer agents. Among the seven synthesized molecules, compounds 6, 8, and 10 exhibited anticancer activity with IC50 values of 19.98 ± 3.38, 38.23 ± 3.30, and 46.95 ± 5.68 µM, respectively, against human colorectal adenocarcinoma (HT-29) cells. Clonogenic assays and Western blot analysis tests further confirmed that compound 6 effectively induced apoptosis in HT-29 cells through mitochondrial- and caspase-3-dependent pathways.

Construction of interferon alpha/beta receptor subunit 1 gene knockout Caco-2 cell line based on CRISPR/Cas9 system / 中国生物制品学杂志

@#Objective To knockout interferon alpha/beta receptor subunit 1(IFNAR1) gene in human colorectal adenocarcinoma cells Caco-2 using clustered regularly interspaced short palinmic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system to construct IFNAR1 knockout Caco-2 cell line.Methods The single guide RNA(sgRNA)sequence was designed to specifically recognize the exon region of IFNAR1 gene using CRISPR/Cas9 technology,and the LentiCRISPRv2-IFNAR1-sgRNA recombinant plasmid was constructed.Caco-2 cells were infected with the plasmid packaged by lentivirus and screened by puromycin resistance.The obtained monoclonal cell lines were cultured by limited dilution method,which were verified for the effect of IFNAR1 gene knockout by target gene sequencing and Western blot,and detected for the mRNA levels of CXC chemokine ligand 10(CXCL10)and interferon-stimulatd gene 20(ISG20)in IFNAR1knockout cells by adding exogenous IFNβ.Results Sequencing results of plasmid LentiCRISPRv2-IFNAR1-sgRNA showed that the insertion sites were all located at the sticky end of BsmBⅠenzyme digestion.Two IFNAR1 knockout monoclonal cell lines were obtained.The sequencing results showed that Caco-2-IFNAR1-KO1 had 5 bp deletion in the sixth exon of IFNAR1,and Caco-2-IFNAR1-KO2 had 18 bp deletion and 1 bp insertion in the seventh exon.Compared with wild-type Caco-2 cells,Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells showed no expression of IFNAR1 protein.Compared with no IFNβ stimulation,the mRNA levels of CXCL10 gene(t = 0.566 and 1.268 respectively,P>0.05)and ISG20 gene(t =1.522 and 1.733 respectively,P>0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells stimulated by 50 ng/mL IFNβ showed no significant increase.While compared with those of wild-type Caco-2 cells,the mRNA levels of CXCL10gene(t = 6.763 and 6.777 respectively,P<0.05)and ISG20 gene(t = 5.664 and 5.65 respectively,P<0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells decreased significantly under the stimulation of 50 ng/mL exogenous IFNβ.Conclusion Caco-2 cell line with IFNAR1 knockout was successfully constructed by using CRISPR/Cas9 technology,and the downstream molecules activated by IFNAR(interferon alpha/beta receptor)in this cell line were obviously inhibited,which provided a powerful tool for further exploration of the innate immune response and replication packaging mechanism of Caco-2 cells after virus infection.

Extracellular Signals of a Human Epithelial Colorectal Adenocarcinoma (Caco-2) Cell Line Facilitate the Penetration of Pseudomonas aeruginosa PAO1 Strain through the Mucin Layer.

Pseudomonas aeruginosa can penetrate the layer of mucus formed by host intestinal epithelial cells, often resulting in sepsis in immunocompromised patients. We have previously demonstrated that P. aeruginosa can penetrate the mucin layer by flagellar motility and the degradation of the mucin layer. However, it remains unclear how P. aeruginosa initially recognizes epithelial cells. Using the human epithelial colorectal adenocarcinoma (Caco-2) cell line, we investigated extracellular signaling that could facilitate the penetration of P. aeruginosa through the mucin layer. The supernatant from Caco-2 cell cultures increased penetration of P. aeruginosa through an artificial mucin layer. The Caco-2 cell supernatant increased bacterial flagella-dependent swarming motility, but it did not influence P. aeruginosa growth or protease activity. Filtering of the Caco-2 cell supernatant indicated that proteins weighing <10 kDa increased mucin penetration, swarming motility, and, based on a tethered cell assay, induced acceleration of the flagellar filament rotational rate. Furthermore, a capillary assay showed that <10 kDa proteins in the Caco-2 cell supernatant attracted P. aeruginosa cells. Finally, we identified that growth-regulated oncogene-α (GRO-α) secreted by Caco-2 cells was a factor facilitating flagellar filament rotation and swarming motility, although it did not attract the bacteria. We conclude that penetration of the mucin layer by P. aeruginosa is facilitated by small proteins (<10 kDa) secreted by Caco-2 cells, both by inducing acceleration of flagellar motility and increasing chemotaxis.