Ethanol exposure exacerbates 4-nitroquinoline-1-oxide induced esophageal carcinogenesis and induces invasive carcinoma with muscularis propria infiltration in a mouse model.

Esophageal squamous cell carcinoma (ESCC) is one of the most fatal cancers worldwide. Most ESCC patients are diagnosed at an advanced stage; however, current research on in vivo animal models accurately reflecting their clinical presentation is lacking. Alcohol consumption is a major risk factor for ESCC and has been used in several disease models for disease induction. In this study, we used 4-nitroquinoline-1-oxide in combination with ethanol to induce an in vivo ESCC mouse model. Esophageal tissues were stained with hematoxylin and eosin for histopathological examination and lesion scoring. In cellular experiments, cell adhesion and migration invasion ability were observed using phalloidin staining, cell scratch and transwell assays, respectively, and the expression of epithelial-mesenchymal transition-related markers was detected using quantitative reverse transcription polymerase chain reaction and western blotting. The results showed that ethanol-exposed mice lost more weight and had an increased number of esophageal nodules. Histological examination revealed that the lesion scores of the ethanol-exposed esophageal samples were significantly higher than those of the unexposed esophageal samples. Furthermore, ethanol-exposed esophageal cancer samples had more severe lesions with infiltration of tumor cells into the muscularis propria. In vitro cellular experiments showed that ethanol exposure induced cytoskeletal microfilament formation, promoted cell migration invasion elevated the expression of N-cadherin and Snail, and decreased the expression of E-cadherin. In conclusion, ethanol exposure exacerbates ESCC, promotes tumor cell infiltration into the muscularis propria, and could be an effective agent for establishing innovative models of invasive carcinoma.

Bioinformatics and network pharmacology identify promotional effects and potential mechanisms of ethanol on esophageal squamous cell carcinoma and experimental validation.

Ethanol is an important risk factor for esophageal squamous cell carcinoma (ESCC); however, the molecular mechanisms behind how ethanol promotes ESCC development remain poorly understood. In this study, ethanol-ESCC-associated target genes were constructed and screened using network pharmacology and subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) and bioinformatics analysis. A mouse ethanol-exposed esophageal cancer model was constructed with 4-nitroquinoline-1-oxide (4-NQO) to assess its survival and tumor lesion status, and the mechanism of ethanol-promoted ESCC lesions was verified by qRT-PCR and Western blotting. The results showed that 126 ethanol-ESCC crossover genes were obtained, which were significantly enriched in the PI3K/AKT signaling pathway. Bioinformatics results showed that the target genes TNF, IL6, IL1ß and JUN were highly expressed in esophageal tumor samples and positively correlated with tumor proliferation and apoptosis genes, and the genetic information of these genes was mutated to different degrees. Animal model experiments showed that ethanol decreased the survival rate and aggravated the occurrence of esophageal cancer in mice. qRT-PCR showed that ethanol promoted the expression of TNF, IL6, IL1ß and JUN mRNA in mouse esophageal tumor tissues, and Western blotting showed that ethanol promoted p-PI3K and p-AKT protein expression in mouse esophageal tumor tissues. In conclusion, ethanol promotes esophageal carcinogenesis by increasing the expression of TNF, IL6, IL1ß and JUN and activating the PI3K/AKT signaling pathway.

Network Pharmacology Analysis and In Vitro Validation of the Active Ingredients and Potential Mechanisms of Gynostemma Pentaphyllum Against Esophageal Cancer.

BACKGROUND: Esophageal cancer (EC) is one of the deadliest malignancies worldwide. Gynostemma pentaphyllum Thunb. Makino (GpM) has been used in traditional Chinese medicine as a treatment for tumors and hyperlipidemia. Nevertheless, the active components and underlying mechanisms of anti-EC effects of GpM remain elusive. OBJECTIVE: This study aims to determine the major active ingredients of GpM in the treatment of EC and to explore their molecular mechanisms by using network pharmacology, molecular docking, and in vitro experiments. METHODS: Firstly, active ingredients and potential targets of GpM, as well as targets of EC, were screened in relevant databases to construct a compound-target network and a protein-protein interaction (PPI) network that narrowed down the pool of ingredients and targets. This was followed by gene ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Next, molecular docking, ADME and toxicity risk prediction, cell viability assays, in vitro scratch assays, Transwell cell invasion assays, and Western blotting analysis were subsequently applied to validate the results of the network analysis. RESULTS: The screening produced a total of 21 active ingredients and 167 ingredient-related targets for GpM, along with 2653 targets for EC. The PPI network analysis highlighted three targets of interest, namely AKT1, TP53, and VEGFA, and the compound-target network identified three possible active ingredients: quercetin, rhamnazin, and isofucosterol. GO and EKGG indicated that the mechanism of action might be related to the PI3K/AKT signaling pathway as well as the regulation of cell motility and cell migration. Molecular docking and pharmacokinetic analyses suggest that quercetin and isoprostanoid sterols may have therapeutic value and safety for EC. The in vitro experiments confirmed that GpM can inhibit EC cell proliferation, migration, and invasion and suppress PI3K and AKT phosphorylation. CONCLUSION: Our findings indicate that GpM exerts its anti-tumor effect on EC by inhibiting EC cell migration and invasion via downregulation of the PI3K/AKT signaling pathway. Hence, we have reason to believe that GpM could be a promising candidate for the treatment of EC.

Bacteroidetes promotes esophageal squamous carcinoma invasion and metastasis through LPS-mediated TLR4/Myd88/NF-κB pathway and inflammatory changes.

Gut microbiota plays a crucial role in gastrointestinal tumors. Additionally, gut microbes influence the progression of esophageal cancer. However, the major bacterial genera that affect the invasion and metastasis of esophageal cancer remain unknown, and the underlying mechanisms remain unclear. Here, we investigated the gut flora and metabolites of patients with esophageal squamous cell carcinoma and found abundant Bacteroides and increased secretion and entry of the surface antigen lipopolysaccharide (LPS) into the blood, causing inflammatory changes in the body. We confirmed these results in a mouse model of 4NQO-induced esophageal carcinoma in situ and further identified epithelial-mesenchymal transition (EMT) occurrence and TLR4/Myd88/NF-κB pathway activation in mouse esophageal tumors. Additionally, in vitro experiments revealed that LPS from Bacteroides fragile promoted esophageal cancer cell proliferation, migration, and invasion, and induced EMT by activating the TLR4/Myd88/NF-κB pathway. These results reveal that Bacteroides are closely associated with esophageal cancer progression through a higher inflammatory response level and signaling pathway activation that are both common to inflammation and tumors induced by LPS, providing a new biological target for esophageal cancer prevention or treatment.

Chinese Medicine Formula Siwu-Yin Inhibits Esophageal Precancerous Lesions by Improving Intestinal Flora and Macrophage Polarization.

Siwu-Yin (SWY), a traditional Chinese medicinal formula, can replenish blood and nourish Yin. It was recorded in ancient Chinese medicine books in treating esophageal dysphagia, which has similar symptoms and prognosis with esophageal precancerous lesions and esophageal cancer. However, its effect has not been established in vivo. This study explores the antiesophageal cancer effect of SWY on rats with esophageal precancerous lesions. By performing 16S rRNA gene sequencing and metabolomics, it was suggested that SWY may improve the composition of intestinal flora of rats by regulating the synthesis and secretion of bile acids. In addition, flow cytometry results showed that SWY treatment modified tumor microenvironment by improving macrophage polarization and therefore inhibiting the occurrence of esophageal precancerous lesions.

Qigefang Inhibits Migration, Invasion, and Metastasis of ESCC by Inhibiting Gas6/Axl Signaling Pathway.

BACKGROUND: In recent years, there is an increasing interest in using Traditional Chinese Medicine (TCM) and their patents for the treatment of cancers. Qigefang (QGF) is a TCM formula and has been used for the treatment of metastatic esophageal cancer in China. However, its therapeutic effect on tumors and its mechanism of action is largely unknown. The aim of this study is to explore the role of QGF in the treatment of metastasis of Esophageal Squamous Cell Carcinoma( ESCC). METHODS: Human esophageal carcinoma cell line KYSE150 was used for this study. CCK-8 assay was used to determine the cytotoxicity of QGF. The KYSE150 cells were treated with QGF to determine its effect on cell migration (cell scratch assay and imaging) and invasion (Transwell system based with Matrigel assay). Western blotting was used to investigate the effect of QGF on relevant molecules of signaling pathways. A mouse model of lung metastasis of esophageal cancer was established by injecting the KYSE150-Luc cells through the tail vein. A small animal imaging system was used to observe tumor metastasis in the mice. RESULTS: QGF reduced cell migration and invasion of KYSE150 cells. QGF significantly inhibited lung metastasis in nude mice. Further study revealed that the expression of Growth arrest-specific 6 (Gas6), Anexelekto (Axl), N-Nuclear factor-kappa B (NF-κB) and matrix metalloproteinase-9 (MMP-9) proteins were decreased both in vitro and in vivo upon treatment with QGF. CONCLUSION: QGF could prevent invasion and metastasis of esophageal cancer by inhibiting the Gas6/Axl signaling pathway.

Qigesan reduces the motility of esophageal cancer cells via inhibiting Gas6/Axl and NF-κB expression.

The present study is mainly to explore the mechanism that how Qigesan (QGS) affects the movement capacity of esophageal cancer (EC) cell. QGS incubates ECA109 and TE1 cell lines and detecting the motility of tumor cells by different experiments. Growth arrest-specific 6 (Gas6) and Anexelekto (Axl) were co-localized, and then detecting Gas6, Axl signaling pathway, and protein expression after QGS intervention. Similarly, Observing the signal localization and protein expression of P-phosphoinositide3-kinases (PI3K), P-AKT protein kinase B (AKT), P-nuclear factor-kappa B (NF-κB), matrix metalloproteinase-2 (MMP2), and matrix metalloproteinase-9 (MMP9). The results showed that the concentration of QGS was less than 200 ug/ml, and the cultured cells did not exceed 24 h, that no obvious cytotoxicity was observed. QGS significantly inhibited the mobility of ECA109 and TE1 cell lines in the concentration-dependent manner. In addition, QGS can regulate the Gas6/Axl pathway, inhibit the formation and localization of the Gas6/Axl complex, and reduce the protein activation of PI3K/AKT, NF-κB, MMP2, and MMP9. Experimental innovation shows that QGS can significantly slow down the mobility of EC cells by regulating the Gas6/Axl complex and downstream signaling pathways, and provides a theoretical basis for the pharmacological effects of QGS in the therapy of EC.