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In the present study, we examined the role of MDM2 in the angiogenesis process and its potential association with the sprouting of endothelial tip cells. To address this, we performed hypoxia-treated gastric cancer cells (HGC-27) to quantitative RT-PCR and Western blot analysis to measure the levels of MDM2 and VEGF-A mRNA and protein expression. Subsequently, we employed siRNA to disrupt MDM2 expression, followed by hypoxia treatment. The expression levels of MDM2 and VEGF-A mRNA and protein were subsequently reassessed. Additionally, ELISA was utilized to quantify the secretion levels of VEGF-A in each experimental group. A conditioned medium derived from HGC-27 cells treated with different agents was employed to assess its influence on the formation of EA.hy926 endothelial tip cells, using various techniques including Transwell plates migration assays, wound healing experiments, vascular formation assays, scanning electron microscopy, and immunofluorescence staining. These findings demonstrated that the in vitro knockdown of MDM2 in the conditioned medium exhibited significant inhibitory effects on endothelial cell migration, wound healing, and vascular formation. Additionally, the intervention led to a reduction in the presence of CD34+ tip cells and the formation of filopodia in endothelial cells, while partially restoring the integrity of tight junctions. Subsequent examination utilizing RNA-seq revealed that the suppression of MDM2 in HGC-27 cells resulted in the downregulation of the PI3K/AKT signaling pathway. Consequently, this downregulation led to an elevation in angiogenic effects induced by hypoxia.
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Background: Previous clinical studies and randomized controlled trials have revealed that low serum vitamin D levels are associated with the risk of developing insulin resistance. Magnesium has been reported to be a protective factor for insulin resistance, and magnesium has been considered an important co-factor for vitamin D activation. However, the effect of dietary magnesium intake on the relationship between vitamin D and the risk of developing insulin resistance has not been comprehensively investigated. Therefore, we designed this cross-sectional analysis to assess whether dietary magnesium intake modifies the association of vitamin D and insulin resistance. Methods: A total of 4,878 participants (male: 48.2%) from 4 consecutive cycles of the National Health and Nutrition Examination Survey (2007-2014) were included in this study after a rigorous screening process. Participants were stratified by their dietary magnesium intake into low-intake (<267 mg/day) and high-intake (≥267 mg/day) groups. We assessed differences between serum vitamin D levels and the risk of developing insulin resistance (interaction test), using a weighted multivariate logistic regression to analyze differences between participants with low and high magnesium intake levels. Results: There was a negative association between vitamin D and insulin resistance in the US adult population [OR: 0.93 (0.88-0.98)], P < 0.001. Dietary magnesium intake strengthened the association (P for interaction < 0.001). In the low dietary magnesium intake group, vitamin D was negatively associated with the insulin resistance [OR: 0.94 (0.90-0.98)]; in the high dietary magnesium intake group, vitamin D was negatively associated with insulin resistance [OR: 0.92 (0.88-0.96)]. Conclusion: Among adults in the United States, we found an independent association between vitamin D level and insulin resistance, and this association was modified according to different levels of magnesium intake.
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BACKGROUND: Photodynamic therapy (PDT) is based on photochemical and photobiological reactions mediated by photosensitizers to achieve a killing effect on diseased cells. It is used in the treatment of malignant tumors, precancerous lesions and infections. OBJECTIVE: In order to provide theoretical data for further study of the mechanism of PDT for colorectal cancer, SW480 cells were treated with Ce6-PDT and effect of photodynamic therapy (Ce6-PDT) on cytoskeleton and E-cadherin protein were observed. METHODS: The survival of SW480 cells was detected by MTT assay. The morphological changes of SW480 cells after Ce6-PDT were observed by scanning electron microscope (ESM). The migration ability was determined by wound healing assay. The distribution of F-actin in the cytoplasm was observed with confocal laser scanning microscope. Western blot analysis was used to detect the expression of cytoskeleton proteins in SW480 cells after Ce6-PDT. RESULTS: Compared with the control group, there was significant difference in cell viability of cells treated with Ce6-PDT (F = 78753.78, P < 0.05). The pseudopodia almost disappeared and cellular atrophy was clearly visible in the cells of Ce6-PDT group. The migration ability of cells treated with Ce6-PDT for 48 h was significantly lower than the control group (F = 11.794, P<0.001). The result of Western blot analysis showed that the expression of F-actin, α-tubulin, ß-tubulin and Vimentin in the cells treated with Ce6-PDT were significantly higher than that in the control group (F = 22.251,8.109, 5.840, 4.685 and 18.754, P < 0.05). The expression of E-cadherin in cells of Ce6-PDT group was significantly higher than that in control group (F = 30.882, P < 0.001). Perhaps Ce6-PDT inhibits the proliferation and migration of colon cancer SW480 cells by enhancing the expression of E-cadherin, causing the disappearance of cell pseudopodia and the destruction of cytoskeleton. CONCLUSIONS: The destruction of cytoskeleton might be one of the reasons for the inhibition of cell proliferation and migration by Ce6-PDT.
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Neoplasias del Colon , Fotoquimioterapia , Porfirinas , Apoptosis , Línea Celular Tumoral , Clorofilidas , Neoplasias del Colon/tratamiento farmacológico , Citoesqueleto , Humanos , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Porfirinas/farmacologíaRESUMEN
BACKGROUND: Colorectal cancer is one of the most common gastrointestinal malignancies. Photodynamic therapy (PDT) is a novel and non-invasive treatment for tumors as PDT features small trauma, good applicability, andaccurate targeting. PDT may also be a potential treatment for colon cancer as itmay may induce suppressive effects on metastatic potential.. However, the molecular mechanism of the Chlorin e6 Photodynamic therapy (Ce6-PDT) inhibiting the migration of human colon cancer SW620 cells remains unclear. METHODS: Scratch wound healing assay, scanning electron microscope, MTT, immunofluorescence and laser confocal technique were used to investigate the suppressive effects of Ce6-PDT on the SW620 cells migration, pseudopodia, viability and the actin cytoskeleton. The effect of Ce6-PDT on actin-Filaments and signaling molecules of the Rac1/PAK1/LIMK1/cofilin signaling pathway in SW620 cells were examined by western blot analysis. RNA interference (RNAi) technology was used to establish siRNA-Rac1/SW620 cells. The combined effects of Ce6-PDT and RNAi on colon cancer SW620 cells was investigated by the same technology and methods mentioned above to clarify the signal transduction effect of Rac1/PAK1/LIMK1/cofilin signaling pathway in Ce6-PDT caused inhibition of SW620 cell migration. RESULTS: The healing and migration rate of the SW620 cells was significantly reduced and the cell pseudopodia were reduced or disappeared by Ce6-PDT. The Immunofluorescence and western blot analysis results showed that Ce6-PDT destroy microfilament's original structure and significantly downregulated F-actin protein expression. The Rac1/PAK1/LIMK1/cofilin signaling pathway was downregulated by Ce6-PDT. Furthermore, the RNAi significantly strengthened the effect of Ce6-PDT on colon cancer SW620 cells migration. CONCLUSIONS: Actin cytoskeleton and protrusions of SW620 cells correlate with its migration ability. Ce6-PDT suppresses SW620 cells migration by downregulating the Rac1/PAK1/LIMK1/cofilin signaling pathway, and its suppressive effect was enhanced by knocking down Rac1 gene expression.